U.S. patent application number 15/490533 was filed with the patent office on 2017-08-10 for heterocycle-substituted pyridyl benzothiophenes as kinase inhibitors.
The applicant listed for this patent is Allergan, Inc.. Invention is credited to Sougato Boral, Thomas C. Malone, Sandhya Rao, Shimiao Wang, Rong Yang.
Application Number | 20170226094 15/490533 |
Document ID | / |
Family ID | 55654997 |
Filed Date | 2017-08-10 |
United States Patent
Application |
20170226094 |
Kind Code |
A1 |
Boral; Sougato ; et
al. |
August 10, 2017 |
HETEROCYCLE-SUBSTITUTED PYRIDYL BENZOTHIOPHENES AS KINASE
INHIBITORS
Abstract
This invention is directed to a compound of Formula I
##STR00001## or a pharmaceutically acceptable salt thereof, wherein
R.sup.1, R.sup.2, R.sup.3, R.sup.4 and X are as defined herein. The
compounds of Formula I are useful as receptor tyrosine kinase (RTK)
inhibitors and can be used to treat such diseases as cancer, blood
vessel proliferative disorders, fibrotic disorders, mesangial cell
proliferative disorders and metabolic diseases.
Inventors: |
Boral; Sougato; (Irvine,
CA) ; Malone; Thomas C.; (Irvine, CA) ; Wang;
Shimiao; (Tustin, CA) ; Rao; Sandhya; (Irvine,
CA) ; Yang; Rong; (Mission Viejo, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Allergan, Inc. |
Irvine |
CA |
US |
|
|
Family ID: |
55654997 |
Appl. No.: |
15/490533 |
Filed: |
April 18, 2017 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15152404 |
May 11, 2016 |
9650366 |
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|
15490533 |
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|
14511004 |
Oct 9, 2014 |
9359336 |
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15152404 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07D 409/14 20130101;
C07D 405/14 20130101; C07D 401/04 20130101 |
International
Class: |
C07D 409/14 20060101
C07D409/14 |
Claims
1. A compound of Formula I: ##STR00055## or a pharmaceutically
acceptable salt thereof, wherein: R is selected from the group
consisting of: ##STR00056##
2. The compound of claim 1, wherein the compound is
1-[4-chloro-3-(trifluoromethyl)phenyl]-3-{2-[5-(2H-tetrazol-5-yl)pyridin--
3-yl]-1-benzothien-5-yl}urea, or a pharmaceutically acceptable salt
thereof.
3. The compound of claim 1, wherein the compound is
1-(2-{2-amino-5-[1-(3-hydroxypropyl)-1H-tetrazol-5-yl]pyridin-3-yl}-1-ben-
zothien-5-yl)-3-[4-chloro-3-(trifluoromethyl)phenyl]urea, or a
pharmaceutically acceptable salt thereof.
4. The compound of claim 1, wherein the compound is
1-[2-(2-amino-5-pyrimidin-2-ylpyridin-3-yl)-1-benzothien-5-yl]-3-[4-chlor-
o-3-(trifluoromethyl)phenyl]urea, or a pharmaceutically acceptable
salt thereof.
5. The compound of claim 1, wherein the compound is
1-[2-(2-amino-5-pyrazin-2-ylpyridin-3-yl)-1-benzothien-5-yl]-3-[4-chloro--
3-(trifluoromethyl)phenyl]urea, or a pharmaceutically acceptable
salt thereof.
6. A pharmaceutical composition comprising as active ingredient a
therapeutically effective amount of a compound according to claim 1
and a pharmaceutically acceptable adjuvant, diluent or carrier.
7. A pharmaceutical composition comprising as active ingredient a
therapeutically effective amount of a compound according to claim 2
and a pharmaceutically acceptable adjuvant, diluent or carrier.
8. A pharmaceutical composition comprising as active ingredient a
therapeutically effective amount of a compound according to claim 3
and a pharmaceutically acceptable adjuvant, diluent or carrier.
9. A pharmaceutical composition comprising as active ingredient a
therapeutically effective amount of a compound according to claim 4
and a pharmaceutically acceptable adjuvant, diluent or carrier.
10. A pharmaceutical composition comprising as active ingredient a
therapeutically effective amount of a compound according to claim 5
and a pharmaceutically acceptable adjuvant, diluent or carrier.
Description
CROSS-RELATED APPLICATIONS
[0001] This application is a continuation of U.S. patent
application Ser. No. 15/152,404, filed May 11, 2016, which is a
continuation of U.S. patent application Ser. No. 14/511,004, filed
Oct. 9, 2014, now U.S. Pat. No. 9,359,336, issued Jun. 7, 2016, the
disclosures of which are hereby incorporated by reference in their
entireties and serve as the basis of a priority and/or benefit
claim for the present application.
FIELD OF THE INVENTION
[0002] The invention relates to inhibitors of vascular endothelial
growth factor receptor 2 kinase (VEGFR2) or VEGFR, platelet derived
growth factor beta (PDGFR.beta.) kinases or PDGFR and Protein
Kinase R (EIF2AK2), and methods of using such compounds. The
present invention is also directed to methods of regulating,
modulating or inhibiting protein kinases, whether of the receptor
or non-receptor class, for the prevention and/or treatment of
disorders related to unregulated protein kinase signal
transduction, including cell growth, metabolic, blood vessel
proliferative, inflammatory and neurodegenerative disorders.
DESCRIPTION OF THE RELATED ART
[0003] Protein kinases (PKs) comprise a large and diverse class of
proteins having enzymatic activity which catalyzes the transfer of
the terminal phosphate of ATP to the hydroxyl group of a serine,
threonine or tyrosine group in a protein. Protein kinases (PKs) are
involved in numerous diseases which result from dysregulation of
their normal function.
[0004] There are numerous examples where protein kinases, have been
found to be involved in cellular signaling pathways leading to
pathological conditions. In the VEGFR2 kinase protein kinase, which
is a receptor tyrosine kinase, pathological conditions involving
aberrant angiogenesis include cancer, wet age-related macular
degeneration (Ni et al. Ophthalmologica 2009 223 401-410; Chappelow
et al. Drugs 2008 68 1029-1036), diabetic retinopathy (Zhang et al
Int. J. Biochem. Cell Biol. 2009 41 2368-2371), cancer (Aora et al.
J. Path. Exp. Ther. 2006, 315, 971), psoriasis (Heidenreich et al
Drug News Perspective 2008 21 97-105) and hyper immune response. In
ophthalmic diseases such as neovascular age-related macular
degeneration and diabetic retinopathy aberrant activation of VEGF
receptors can lead to abnormal blood vessel growth. The importance
of VEGFR signaling in the neovascular age-related macular
degeneration disease process is evident by the clinical success of
multiple anti-VEGF targeting agents including Lucentis.RTM.,
Avastin.RTM., and EYLEA.TM. (Barakat et al. Expert Opin. Investig.
Drugs 2009, 18, 637). Recently it has been suggested that
inhibition of multiple protein kinase signaling pathways may
provide a greater therapeutic effect than targeting a single
signaling pathway. For example in neovascular ocular disorders such
as neovascular age-related macular degeneration and diabetic
retinopathy the inhibition of both VEGFR and PDGFR.beta. may
provide a greater therapeutic effect in by causing regression of
existing neovascular blood vessels present in the disease (Adamis
et al. Am. J. Pathol. 2006 168 2036-2053). In cancer inhibition of
multiple PK signaling pathways has been suggested to have a greater
effect than inhibiting a single PK pathway (DePinho et al. Science
2007 318 287-290; Bergers et al. J. Clin Invest. 2003 111
1287-1295).
[0005] It has also been suggested that misregulated protein kinases
are involved in neurodegenerative disease. In particular Protein
Kinase R has been implicated in neurodegenerative disease. Protein
Kinase R (PKR, also known as interferon-induced, double-stranded
RNA-activated protein kinase, or eukaryotic translation initiation
factor 2-alpha kinase 2) is one of four known mammalian kinases
that phosphorylate eukaryotic translation initiation factor 2-alpha
(elF-2.alpha.) in response to a variety of stress conditions
(Donnelly et al., Cell. Mol. Life Sci. 2013, 70, 3493-3511). PKR
plays a central role in the innate immune system and serves to
prevent viral replication and viral infection (for a detailed
review see Garcia et al., Microbiol. and Mol. Bio. Rev. 2006, 70,
1032-1060). It is proposed that in chronic conditions like AMD,
innate immune players respond to modified host derived elements
(ROS/Alu) and external particulate matter (drusen) by activation of
inflammasome complex. Emerging evidence indicates that PKR has a
key role in NLRP3 inflammasome activation (Yim & Williams; J of
Interferon & Cytokine Res, 2014, Campbell & Doyle, J Mol
Med, 2013, Lu et. al; Nature, 2012).
[0006] The binding of double stranded RNA to the double stranded
RNA regulatory domains of PKR induces dimerization and
autophosphorylation which leads to activation of the kinase (Dever
et al., Cell 2005, 122, 901-913). Once activated by dimerization
PKR can suppress protein synthesis by phosphorylation of serine-51
on eukaryotic translation initiation factor 2-alpha (elF-2.alpha.).
In its phosphorylated form elF2alpha increases its affinity for
elF-2B by 100-fold effectively converting it into a competitive
inhibitor of elF-2B. By this mechanism a small amount of
phosphorylated elF2alpha can effectively inhibit the guanine
nucleotide exchange activity of elF-2B and shut down protein
translation (Ramaiah et al., Biochemistry 2000, 39,
12929-12938).
[0007] In addition to PKR's role in regulation of protein synthesis
it also plays an important role in signal transduction linked to
apoptotic cell death. PKR has been shown to be activated by dsRNA,
number of growth factors and cytokines including INF, PDGF,
TNF-alpha, and IL-1 and by the activation of Toll receptors. PKR
has also been shown to be phosphorylated by JAK1 and Tyk2 kinases
(Su et al., EMBO Reports 2007, 3, 265). Activation of PKR leads to
the activation of multiple signaling pathways that are involved in
inflammation and cell death. PKR is required for phosphorylation of
MKK6 (Williams et al., J. Biol. Chem. 2004, 279, 37670-37676) and
subsequent p38 MAPK signaling (Williams et al., The EMBO Journal
2000, 19, 4292-4297). PKR induces the expression of the pro
apoptotic factor CHOP and has been shown to induce apoptosis by the
FADD/Caspase 8 pathway (Barber, G. et al, The EMBO Journal 1998,
17, 6888-6902).
[0008] Due to its key role in regulation of apoptotic cell death
PKR inhibition may be useful in prevention of the rod and cone
photoreceptor cell death and ganglion cell death associated with
the atrophic form of macular degeneration (Shimazawa et al, IVOS
2007, 48, 3729-3736).
[0009] The identification of effective small compounds which
specifically inhibit signal transduction by modulating the activity
of receptor and non-receptor protein kinases to regulate and
modulate abnormal or inappropriate cell proliferation is therefore
desirable and one object of this invention.
[0010] Certain small compounds are disclosed in PCT publication No.
WO/1999/062890, PCT publication No. WO/2005/082001 and PCT
publication No. WO/2006/026034 as useful for the treatment of
diseases related to unregulated TKS transduction.
SUMMARY OF THE INVENTION
[0011] The present invention relates to organic molecules capable
of modulating, regulating and/or inhibiting tyrosine kinase signal
transduction by blocking the VEGF and/or PDGF receptors. Such
compounds are useful for the treatment of diseases related to
unregulated PTKs transduction, including cell proliferative
diseases such as cancer; vascular (blood vessel) proliferative
disorders such as mesangial cell proliferative disorders and
metabolic diseases, lung carcinomas, breast carcinomas, Non
Hodgkin's lymphomas, ovarian carcinoma, pancreatic cancer,
malignant pleural mesothelioma, melanoma, arthritis, restenosis,
hepatic cirrhosis, atherosclerosis, psoriasis, rosacea, diabetic
mellitus, wound healing and inflammation and preferably ophthalmic
diseases, i.e. diabetic retinopathy, retinopathy of prematurity,
macular edema, retinal vein occlusion, exudative or neovascular
age-related macular degeneration, high-risk eyes (i.e. fellow eyes
have neovascular age-related macular degeneration) with dry
age-related macular degeneration, neovascular disease associated
with retinal vein occlusion, neovascular disease (including
choroidal neovascularization) associated with the following:
pathologic myopia, pseudoxanthoma elasticum, optic nerve drusen,
traumatic choroidal rupture, central serous retinopathy, cystoid
macular edema, diabetic retinopathy, proliferative diabetic
retinopathy, diabetic macular edema, rubeosis iridis, retinopathy
of prematurity, Central and branch retinal vein occlusions,
inflammatory/infectious retinal, neovascularization/edema, corneal
neovascularization, hyperemia related to an actively inflamed
pterygia, recurrent pterygia following excisional surgery,
post-excision, progressive pterygia approaching the visual axis,
prophylactic therapy to prevent recurrent pterygia, of
post-excision, progressive pterygia approaching the visual axis,
chronic low grade hyperemia associated with pterygia, neovascular
glaucoma, iris neovascularization, idiopathic etiologies, presumed
ocular histoplasmosis syndrome, retinopathy of prematurity, chronic
allergic conjunctivitis, ocular rosacea, blepharoconjunctivitis,
recurrent episcleritis, keratoconjunctivitis sicca, ocular graft vs
host disease, etc.
[0012] In one aspect, the invention provides a compound represented
by Formula I or a pharmaceutically acceptable salt thereof or
stereoisomeric forms thereof, or the enantiomers, diastereoisomers,
tautomers, zwitterions and pharmaceutically acceptable salts
thereof:
##STR00002##
wherein:
[0013] R.sup.1 is hydrogen, substituted or unsubstituted
C.sub.1-C.sub.8 alkyl;
[0014] X is
##STR00003##
[0015] R.sup.2 is substituted or unsubstituted heterocycle,
##STR00004##
or substituted or unsubstituted aryl;
[0016] R.sup.3 is substituted or unsubstituted heterocycle,
substituted or unsubstituted aryl,
[0017] R.sup.4 is hydrogenor substituted or unsubstituted
C.sub.1-C.sub.8 alkyl; and
[0018] R.sup.5 is substituted or unsubstituted heterocycle or
substituted or unsubstituted aryl.
[0019] In another aspect, the invention provides a compound
represented by Formula I wherein:
[0020] R.sup.1 is hydrogen, substituted or unsubstituted
C.sub.1-C.sub.8 alkyl;
[0021] X is
##STR00005##
[0022] R.sup.2 is substituted or unsubstituted heterocycle,
##STR00006##
or substituted or unsubstituted aryl;
[0023] R.sup.3 is substituted or unsubstituted heterocycle,
substituted or unsubstituted aryl,
[0024] R.sup.4 is hydrogenor substituted or unsubstituted
C.sub.1-C.sub.8 alkyl; and
[0025] R.sup.5 is substituted or unsubstituted heterocycle or
substituted or unsubstituted aryl.
In another aspect, the invention provides a compound represented by
Formula I wherein:
[0026] R.sup.1 is hydrogen, substituted or unsubstituted
C.sub.1-C.sub.8 alkyl;
[0027] X is
##STR00007##
[0028] R.sup.2 is substituted or unsubstituted heterocycle,
##STR00008##
or substituted or unsubstituted aryl;
[0029] R.sup.3 is substituted or unsubstituted heterocycle,
substituted or unsubstituted aryl,
[0030] R.sup.4 is hydrogenor substituted or unsubstituted
C.sub.1-C.sub.8 alkyl; and
[0031] R.sup.5 is substituted or unsubstituted heterocycle or
substituted or unsubstituted aryl.
[0032] In another aspect, the invention provides a compound
represented by Formula I wherein:
[0033] R.sup.1 is hydrogen, substituted or unsubstituted
C.sub.1-C.sub.8 alkyl;
[0034] X is
##STR00009##
[0035] R.sup.2 is substituted or unsubstituted heterocycle,
##STR00010##
or substituted or unsubstituted aryl;
[0036] R.sup.3 is substituted or unsubstituted heterocycle,
substituted or unsubstituted aryl,
[0037] R.sup.4 is hydrogenor substituted or unsubstituted
C.sub.1-C.sub.8 alkyl; and
[0038] R.sup.5 is substituted or unsubstituted heterocycle or
substituted or unsubstituted aryl.
[0039] In another aspect, the invention provides a compound
represented by Formula I wherein:
R.sup.1 is hydrogen;
[0040] X is
##STR00011##
[0041] R.sup.2 is substituted or unsubstituted heterocycle;
[0042] R.sup.3 is substituted or unsubstituted aryl,
[0043] R.sup.4 is hydrogen or substituted or unsubstituted
C.sub.1-C.sub.8 alkyl.
[0044] In another aspect, the invention provides a compound
represented by Formula I wherein:
[0045] R.sup.1 is hydrogen;
[0046] X is
##STR00012##
[0047] R.sup.2 is substituted or unsubstituted heterocycle;
[0048] R.sup.3 is substituted or unsubstituted aryl,
[0049] R.sup.4 is hydrogen.
[0050] In another aspect, the invention provides a compound
represented by Formula I wherein:
[0051] R.sup.1 is hydrogen;
[0052] X is
##STR00013##
[0053] R.sup.2 is substituted or unsubstituted heterocycle;
[0054] R.sup.3 is substituted or unsubstituted aryl,
[0055] R.sup.4 is substituted or unsubstituted C.sub.1-C.sub.8
alkyl.
[0056] In another aspect, the invention provides a compound
represented by Formula I wherein:
[0057] R.sup.1 is hydrogen;
[0058] X is
##STR00014## [0059] R.sup.2 is
##STR00015##
[0060] R.sup.3 is substituted or unsubstituted aryl,
[0061] R.sup.4 is substituted or unsubstituted C.sub.1-C.sub.8
alkyl; and
[0062] R.sup.5 is substituted or unsubstituted aryl.
[0063] In another aspect, the invention provides a compound
represented by Formula I wherein:
[0064] R.sup.1 is hydrogen;
[0065] X is H
##STR00016##
[0066] R.sup.2 is
##STR00017##
[0067] R.sup.3 is substituted or unsubstituted heterocycle,
substituted or unsubstituted aryl,
[0068] R.sup.4 is substituted or unsubstituted C.sub.1-C.sub.8
alkyl; and
[0069] R.sup.5 is substituted or unsubstituted aryl.
[0070] In another aspect, the invention provides a compound
represented by Formula I wherein: [0071] R.sup.1 is hydrogen;
[0072] X is H
##STR00018##
[0073] R.sup.2 is substituted or unsubstituted heterocycle;
[0074] R.sup.3 is substituted or unsubstituted heterocycle,
substituted or unsubstituted aryl; and
[0075] R.sup.4 is substituted or unsubstituted C.sub.1-C.sub.8
alkyl.
[0076] The term "alkyl", as used herein, refers to saturated,
monovalent or divalent hydrocarbon moieties having linear or
branched moieties or combinations thereof and containing 1 to 8
carbon atoms. One methylene (--CH.sub.2--) group, of the alkyl
group can be replaced by oxygen, sulfur, sulfoxide, nitrogen,
carbonyl, carboxyl, sulfonyl, sulfate, sulfonate, amide,
sulfonamide, by a divalent C.sub.3-8 cycloalkyl, by a divalent
heterocycle, or by a divalent aryl group. Alkyl groups can have one
or more chiral centers. Alkyl groups can be independently
substituted by halogen atoms, hydroxyl groups, cycloalkyl groups,
amino groups, heterocyclic groups, aryl groups, carboxylic acid
groups, phosphonic acid groups, sulphonic acid groups, phosphoric
acid groups, nitro groups, amide groups, sulfonamide groups, ester
groups, ketone groups.
[0077] The term "cycloalkyl", as used herein, refers to a
monovalent or divalent group of 3 to 8 carbon atoms derived from a
saturated cyclic hydrocarbon. Cycloalkyl groups can be monocyclic
or polycyclic. Cycloalkyl can be independently substituted by
halogen atoms, sulfonyl C.sub.1-8 alkyl groups, sulfoxide C.sub.1-8
alkyl groups, sulfonamide groups, nitro groups, cyano groups,
--OC.sub.1-8 alkyl groups, --SC.sub.1-8 alkyl groups, --C.sub.1-8
alkyl groups, --C.sub.2-6 alkenyl groups, --C.sub.2-6 alkynyl
groups, ketone groups, alkylamino groups, amino groups, aryl
groups, C.sub.3-8 cycloalkyl groups or hydroxyl groups.
[0078] The term "cycloalkenyl", as used herein, refers to a
monovalent or divalent group of 3 to 8 carbon atoms derived from a
saturated cycloalkyl having at least one double bond. Cycloalkenyl
groups can be monocyclic or polycyclic. Cycloalkenyl groups can be
independently substituted by halogen atoms, sulfonyl groups,
sulfoxide groups, nitro groups, cyano groups, --OC.sub.1-6 alkyl
groups, --SC.sub.1-6 alkyl groups, --C.sub.1-6 alkyl groups,
--C.sub.2-6 alkenyl groups, --C.sub.2-6 alkynyl groups, ketone
groups, alkylamino groups, amino groups, aryl groups, C.sub.3-8
cycloalkyl groups or hydroxyl groups.
[0079] The term "halogen", as used herein, refers to an atom of
chlorine, bromine, fluorine, iodine.
[0080] The term "alkenyl", as used herein, refers to a monovalent
or divalent hydrocarbon radical having 2 to 6 carbon atoms, derived
from a saturated alkyl, having at least one double bond. One
methylene (--CH.sub.2--) group, of the alkenyl can be replaced by
oxygen, sulfur, sulfoxide, nitrogen, carbonyl, carboxyl, sulfonyl,
sulfate, sulfonate, amide, sulfonamide, by a divalent C.sub.3-8
cycloalkyl, by a divalent heterocycle, or by a divalent aryl group.
C.sub.2-6 alkenyl can be in the E or Z configuration. Alkenyl
groups can be substituted by alkyl groups, as defined above or by
halogen atoms.
[0081] The term "alkynyl", as used herein, refers to a monovalent
or divalent hydrocarbon radical having 2 to 6 carbon atoms, derived
from a saturated alkyl, having at least one triple bond. One
methylene (--CH.sub.2--) group, of the alkynyl can be replaced by
oxygen, sulfur, sulfoxide, nitrogen, carbonyl, carboxyl, sulfonyl,
sulfate, sulfonate, amide, sulfonamide, by a divalent C.sub.3-8
cycloalkyl, by a divalent heterocycle, or by a divalent aryl group.
Alkynyl groups can be substituted by alkyl groups, as defined
above, or by halogen atoms.
[0082] The term "heterocycle" as used herein, refers to a 3 to 10
membered ring, which can be aromatic or non-aromatic, saturated or
unsaturated, containing at least one heteroatom selected form
oxygen, nitrogen, sulfur, or combinations of at least two thereof,
interrupting the carbocyclic ring structure. The heterocyclic ring
can be interrupted by a C.dbd.O; the S and N heteroatoms can be
oxidized. Heterocycles can be monocyclic or polycyclic.
Heterocyclic ring moieties can be substituted by halogen atoms,
sulfonyl groups, sulfoxide groups, nitro groups, cyano groups,
--OC.sub.1-6 alkyl groups, --SC.sub.1-6 alkyl groups, --C.sub.1-8
alkyl groups, --C.sub.2-6 alkenyl groups, --C.sub.2-6 alkynyl
groups, ketone groups, alkylamino groups, amide groups, amino
groups, aryl groups, C.sub.3-8 cycloalkyl groups or hydroxyl
groups. Examples of heterocycles are thiophene, benzothiophene,
furan, benzofuran, indole.
[0083] The term "aryl" as used herein, refers to an organic moiety
derived from an aromatic hydrocarbon consisting of a ring
containing 6 to 10 carbon atoms, by removal of one hydrogen atom.
Aryl can be substituted by halogen atoms, sulfonyl C.sub.1-6 alkyl
groups, sulfoxide C.sub.1-6 alkyl groups, sulfonamide groups,
carboxyclic acid groups, C.sub.1-6 alkyl carboxylates (ester)
groups, amide groups, nitro groups, cyano groups, --OC.sub.1-6
alkyl groups, --SC.sub.1-6 alkyl groups, --C.sub.1-6 alkyl groups,
--C.sub.2-6 alkenyl groups, --C.sub.2-6 alkynyl groups, ketone
groups, aldehydes, alkylamino groups, amino groups, aryl groups,
C.sub.3-8 cycloalkyl groups or hydroxyl groups. Aryls can be
monocyclic or polycyclic.
[0084] The term "hydroxyl" as used herein, represents a group of
formula "--OH".
[0085] The term "carbonyl" as used herein, represents a group of
formula "--C(O)--".
[0086] The term "ketone" as used herein, represents an organic
compound having a carbonyl group linked to a carbon atom such as
--C(O)R.sup.x wherein R.sup.x can be alkyl, aryl, cycloalkyl,
cycloalkenyl, heterocycle as defined above.
[0087] The term "ester" as used herein, represents an organic
compound having a carbonyl group linked to a carbon atom such as
--C(O)OR.sup.x wherein R.sup.x can be alkyl, aryl, cycloalkyl,
cycloalkenyl, heterocycle as defined above.
[0088] The term "amine" as used herein, represents a group of
formula "--NR.sup.xR.sup.y", wherein R.sup.x and R.sup.y can be the
same or independently H, alkyl, aryl, cycloalkyl, cycloalkenyl,
heterocycle as defined above.
[0089] The term "carboxyl" as used herein, represents a group of
formula "--C(O)O--".
[0090] The term "sulfonyl" as used herein, represents a group of
formula "--SO.sub.2--".
[0091] The term "sulfate" as used herein, represents a group of
formula "--O--S(O).sub.2--O--".
[0092] The term "sulfonate" as used herein, represents a group of
the formula "--S(O).sub.2--O--".
[0093] The term "carboxylic acid" as used herein, represents a
group of formula "--C(O)OH".
[0094] The term "nitro" as used herein, represents a group of
formula "--NO.sub.2".
[0095] The term "cyano" as used herein, represents a group of
formula "--CN".
[0096] The term "amide" as used herein, represents a group of
formula "--C(O)NR.sup.xR.sup.y," or "NR.sup.xR.sup.yC(O)--,"
wherein R.sup.x and R.sup.y can be the same or independently H,
alkyl, aryl, cycloalkyl, cycloalkenyl, heterocycle as defined
above.
[0097] The term "sulfonamide" as used herein, represents a group of
formula "--S(O).sub.2NR.sup.xR.sup.y" wherein R.sup.x and R.sup.y
can be the same or independently H, alkyl, aryl, cycloalkyl,
cycloalkenyl, heterocycle as defined above.
[0098] The term "sulfoxide" as used herein, represents a group of
formula "--S(O)--".
[0099] The term "phosphonic acid" as used herein, represents a
group of formula "--P(O)(OH).sub.2".
[0100] The term "phosphoric acid" as used herein, represents a
group of formula "--OP(O)(OH).sub.2".
[0101] The term "sulphonic acid" as used herein, represents a group
of formula "--S(O).sub.2OH".
[0102] The formula "H", as used herein, represents a hydrogen
atom.
[0103] The formula "O", as used herein, represents an oxygen
atom.
[0104] The formula "N", as used herein, represents a nitrogen
atom.
[0105] The formula "S", as used herein, represents a sulfur
atom.
Other defined terms are used throughout this specification: "DCE"
refers to dichloroethane "DCM" refers to dichloromethane "DMF"
refers to dimethylformamide "EDC" refers to
1-ethyl-3-(3-dimethylaminopropyl)carbodiimide "PDGF" refers to
platelet derived growth factor "PTKs" refers to protein tyrosine
kinase "RTKs" refers to receptor tyrosine kinase "THF" refers to
tetrahydrofuran "VEGF" refers to vascular endothelial growth factor
"VEGFR" refers to vascular endothelial growth factor receptor
Compounds of the invention are: [0106]
1-[4-chloro-3-(trifluoromethyl)phenyl]-3-{2-[5-(2H-tetrazol-5-yl)pyridin--
3-yl]-1-benzothien-5-yl}urea; [0107]
1-{2-[2-amino-5-(2H-tetrazol-5-yl)pyridin-3-yl]-1-benzothien-5-yl}-3-[4-c-
hloro-3-(trifluoromethyl)phenyl]urea; [0108]
1-(2-{2-amino-5-[2-(3-hydroxypropyl)-2H-tetrazol-5-yl]pyridin-3-yl}-1-ben-
zothien-5-yl)-3-[4-chloro-3-(trifluoromethyl)phenyl]urea; [0109]
1-(2-{2-amino-5-[2-(3-hydroxypropyl)-2H-tetrazol-5-yl]pyridin-3-yl}-1-ben-
zothien-5-yl)-3-[4-fluoro-3-(trifluoromethyl)phenyl]urea; [0110]
1-(2-{2-amino-5-[2-(3-hydroxypropyl)-2H-tetrazol-5-yl]pyridin-3-yl}-1-ben-
zothien-5-yl)-3-[3-fluoro-5-(trifluoromethyl)phenyl]urea; [0111]
1-(2-{2-amino-5-[2-(3-hydroxypropyl)-2H-tetrazol-5-yl]pyridin-3-yl}-1-ben-
zothien-5-yl)-3-[3,5-bis(trifluoromethyl)phenyl]urea; [0112]
1-(2-{2-amino-5-[1-(3-hydroxypropyl)-1H-tetrazol-5-yl]pyridin-3-yl}-1-ben-
zothien-5-yl)-3-[4-chloro-3-(trifluoromethyl)phenyl]urea; [0113]
1-(2-{2-amino-5-[2-(2-hydroxyethyl)-2H-tetrazol-5-yl]pyridin-3-yl}-1-benz-
othien-5-yl)-3-[4-chloro-3-(trifluoromethyl)phenyl]urea; [0114]
N-[3-({5-[2-(3-hydroxypropyl)-2H-tetrazol-5-yl]pyridin-3-yl}ethynyl)pheny-
l]-3-methyl-2-furamide; [0115]
N-[3-({5-[2-(3-hydroxypropyl)-2H-tetrazol-5-yl]pyridin-3-yl}ethynyl)pheny-
l]-3-methylbenzamide; [0116]
1-[3-({2-amino-5-[2-(3-hydroxypropyl)-2H-tetrazol-5-yl]pyridin-3-yl}ethyn-
yl)phenyl]-3-[4-chloro-3-(trifluoromethyl)phenyl]urea; [0117]
1-[2-fluoro-5-(trifluoromethyl)phenyl]-3-[4-({5-[2-(3-hydroxypropyl)-2H-t-
etrazol-5-yl]pyridin-3-yl}ethynyl)phenyl]urea; [0118]
1-[2-(2-amino-5-pyrimidin-2-ylpyridin-3-yl)-1-benzothien-5-yl]-3-[4-chlor-
o-3-(trifluoromethyl)phenyl]urea; [0119]
1-[2-(2-amino-5-pyrazin-2-ylpyridin-3-yl)-1-benzothien-5-yl]-3-[4-chloro--
3-(trifluoromethyl)phenyl]urea.
[0120] Compounds of formula I are useful as protein kinase
inhibitors. As such, compounds of formula I will be useful for
treating diseases related to unregulated protein kinase signal
transduction, for example, cancer, blood vessel proliferative
disorders, fibrotic disorders, inflammatory disorders and
neurodegenerative diseases. In particular, the compounds of the
present invention are useful for treatment of mesangial cell
proliferative disorders and metabolic diseases, lung carcinomas,
breast carcinomas, Non Hodgkin's lymphomas, ovarian carcinoma,
pancreatic cancer, malignant pleural mesothelioma, melanoma,
arthritis, restenosis, hepatic cirrhosis, atherosclerosis,
psoriasis, rosacea, diabetic mellitus, wound healing, inflammation
and neurodegenerative diseases and preferably ophthalmic diseases,
i.e. diabetic retinopathy, retinopathy of prematurity, macular
edema, retinal vein occlusion, exudative or neovascular age-related
macular degeneration, high-risk eyes (i.e. fellow eyes have
neovascular age-related macular degeneration) with dry age-related
macular degeneration, neovascular disease associated with retinal
vein occlusion, neovascular disease (including choroidal
neovascularization) associated with the following: pathologic
myopia, pseudoxanthoma elasticum, optic nerve drusen, traumatic
choroidal rupture, atrophic macular degeneration, geographic
atrophy, central serous retinopathy, cystoid macular edema,
diabetic retinopathy, proliferative diabetic retinopathy, diabetic
macular edema, rubeosis iridis, retinopathy of prematurity, Central
and branch retinal vein occlusions, inflammatory/infectious
retinal, neovascularization/edema, corneal neovascularization,
hyperemia related to an actively inflamed pterygia, recurrent
pterygia following excisional surgery, post-excision, progressive
pterygia approaching the visual axis, prophylactic therapy to
prevent recurrent pterygia, of post-excision, progressive pterygia
approaching the visual axis, chronic low grade hyperemia associated
with pterygia, neovascular glaucoma, iris neovascularization,
idiopathic etiologies, presumed ocular histoplasmosis syndrome,
retinopathy of prematurity, chronic allergic conjunctivitis, ocular
rosacea, blepharoconjunctivitis, recurrent episcleritis,
keratoconjunctivitis sicca, ocular graft vs host disease, etc.
[0121] Some compounds of Formula I and some of their intermediates
may have at least one asymmetric center in their structure. This
asymmetric center may be present in an R or S configuration, said R
and S notation is used in correspondence with the rules described
in Pure Appli. Chem. (1976), 45, 11-13.
[0122] The term "pharmaceutically acceptable salts" refers to salts
or complexes that retain the desired biological activity of the
above identified compounds and exhibit minimal or no undesired
toxicological effects. The "pharmaceutically acceptable salts"
according to the invention include therapeutically active,
non-toxic base or acid salt forms, which the compounds of Formula I
are able to form.
[0123] The acid addition salt form of a compound of Formula I that
occurs in its free form as a base can be obtained by treating the
free base with an appropriate acid such as an inorganic acid, for
example, hydrochloric acid, hydrobromic acid, sulfuric acid,
phosphoric acid, nitric acid and the like; or an organic acid such
as for example, acetic acid, hydroxyacetic acid, propanoic acid,
lactic acid, pyruvic acid, malonic acid, fumaric acid, maleic acid,
oxalic acid, tartaric acid, succinic acid, malic acid, ascorbic
acid, benzoic acid, tannic acid, pamoic acid, citric acid,
methylsulfonic acid, ethanesulfonic acid, benzenesulfonic acid,
formic and the like (Handbook of Pharmaceutical Salts, P. Heinrich
Stahl & Camille G. Wermuth (Eds), Verlag Helvetica Chimica
Acta-Zurich, 2002, 329-345).
[0124] The base addition salt form of a compound of Formula I that
occurs in its acid form can be obtained by treating the acid with
an appropriate base such as an inorganic base, for example, sodium
hydroxide, magnesium hydroxide, potassium hydroxide, calcium
hydroxide, ammonia and the like; or an organic base such as for
example, L-Arginine, ethanolamine, betaine, benzathine, morpholine
and the like. (Handbook of Pharmaceutical Salts, P. Heinrich Stahl
& Camille G. Wermuth (Eds), Verlag Helvetica Chimica
Acta-Zurich, 2002, 329-345).
[0125] Compounds of Formula I and their salts can be in the form of
a solvate, which is included within the scope of the present
invention. Such solvates include for example hydrates, alcoholates
and the like.
[0126] With respect to the present invention reference to a
compound or compounds, is intended to encompass that compound in
each of its possible isomeric forms and mixtures thereof unless the
particular isomeric form is referred to specifically.
[0127] Compounds according to the present invention may exist in
different polymorphic forms. Although not explicitly indicated in
the above formula, such forms are intended to be included within
the scope of the present invention.
[0128] The actual amount of the compound to be administered in any
given case will be determined by a physician taking into account
the relevant circumstances, such as the severity of the condition,
the age and weight of the patient, the patient's general physical
condition, the cause of the condition, and the route of
administration.
[0129] The patient will be administered the compound orally in any
acceptable form, such as a tablet, liquid, capsule, powder and the
like, or other routes may be desirable or necessary, particularly
if the patient suffers from nausea. Such other routes may include,
without exception, transdermal, parenteral, subcutaneous,
intranasal, via an implant stent, intrathecal, intravitreal,
topical to the eye, back to the eye, intramuscular, intravenous,
and intrarectal modes of delivery. Additionally, the formulations
may be designed to delay release of the active compound over a
given period of time, or to carefully control the amount of drug
released at a given time during the course of therapy.
[0130] In another embodiment of the invention, there are provided
pharmaceutical compositions including at least one compound of the
invention in a pharmaceutically acceptable carrier thereof. The
phrase "pharmaceutically acceptable" means the carrier, diluent or
excipient must be compatible with the other ingredients of the
formulation and not deleterious to the recipient thereof.
[0131] Pharmaceutical compositions of the present invention can be
used in the form of a solid, a solution, an emulsion, a dispersion,
a patch, a micelle, a liposome, and the like, wherein the resulting
composition contains one or more compounds of the present
invention, as an active ingredient, in admixture with an organic or
inorganic carrier or excipient suitable for enteral or parenteral
applications. Invention compounds may be combined, for example,
with the usual non-toxic, pharmaceutically acceptable carriers for
tablets, pellets, capsules, suppositories, solutions, emulsions,
suspensions, and any other form suitable for use. The carriers
which can be used include glucose, lactose, gum acacia, gelatin,
mannitol, starch paste, magnesium trisilicate, talc, corn starch,
keratin, colloidal silica, potato starch, urea, medium chain length
triglycerides, dextrans, and other carriers suitable for use in
manufacturing preparations, in solid, semisolid, or liquid form. In
addition auxiliary, stabilizing, thickening and coloring agents and
perfumes may be used. Invention compounds are included in the
pharmaceutical composition in an amount sufficient to produce the
desired effect upon the process or disease condition.
[0132] Pharmaceutical compositions containing invention compounds
may be in a form suitable for oral use, for example, as tablets,
troches, lozenges, aqueous or oily suspensions, dispersible powders
or granules, emulsions, hard or soft capsules, or syrups or
elixirs. Compositions intended for oral use may be prepared
according to any method known in the art for the manufacture of
pharmaceutical compositions and such compositions may contain one
or more agents selected from the group consisting of a sweetening
agent such as sucrose, lactose, or saccharin, flavoring agents such
as peppermint, oil of wintergreen or cherry, coloring agents and
preserving agents in order to provide pharmaceutically elegant and
palatable preparations. Tablets containing invention compounds in
admixture with non-toxic pharmaceutically acceptable excipients may
also be manufactured by known methods. The excipients used may be,
for example, (1) inert diluents such as calcium carbonate, lactose,
calcium phosphate or sodium phosphate; (2) granulating and
disintegrating agents such as corn starch, potato starch or alginic
acid; (3) binding agents such as gum tragacanth, corn starch,
gelatin or acacia, and (4) lubricating agents such as magnesium
stearate, stearic acid or talc. The tablets may be uncoated or they
may be coated by known techniques to delay disintegration and
absorption in the gastrointestinal tract and thereby provide a
sustained action over a longer period. For example, a time delay
material such as glyceryl monostearate or glyceryl distearate may
be employed.
[0133] In some cases, formulations for oral use may be in the form
of hard gelatin capsules wherein the invention compounds are mixed
with an inert solid diluent, for example, calcium carbonate,
calcium phosphate or kaolin. They may also be in the form of soft
gelatin capsules wherein the invention compounds are mixed with
water or an oil medium, for example, peanut oil, liquid paraffin or
olive oil.
[0134] The pharmaceutical compositions may be in the form of a
sterile injectable suspension. This suspension may be formulated
according to known methods using suitable dispersing or wetting
agents and suspending agents. The sterile injectable preparation
may also be a sterile injectable solution or suspension in a
non-toxic parenterally-acceptable diluent or solvent, for example,
as a solution in 1,3-butanediol. Sterile, fixed oils are
conventionally employed as a solvent or suspending medium. For this
purpose any bland fixed oil may be employed including synthetic
mono- or diglycerides, fatty acids (including oleic acid),
naturally occurring vegetable oils like sesame oil, coconut oil,
peanut oil, cottonseed oil, etc., or synthetic fatty vehicles like
ethyl oleate or the like. Buffers, preservatives, antioxidants, and
the like can be incorporated as required.
[0135] Pharmaceutical compositions containing invention compounds
may be in a form suitable for topical use, for example, as oily
suspensions, as solutions or suspensions in aqueous liquids or
nonaqueous liquids, or as oil-in-water or water-in-oil liquid
emulsions. Pharmaceutical compositions may be prepared by combining
a therapeutically effective amount of at least one compound
according to the present invention, or a pharmaceutically
acceptable salt thereof, as an active ingredient with conventional
ophthalmically acceptable pharmaceutical excipients and by
preparation of unit dosage suitable for topical ocular use. The
therapeutically efficient amount typically is between about 0.0001
and about 5% (w/v), preferably about 0.001 to about 2.0% (w/v) in
liquid formulations.
[0136] For ophthalmic application, preferably solutions are
prepared using a physiological saline solution as a major vehicle.
The pH of such ophthalmic solutions should preferably be maintained
between 4.5 and 8.0 with an appropriate buffer system, a neutral pH
being preferred but not essential. The formulations may also
contain conventional pharmaceutically acceptable preservatives,
stabilizers and surfactants. Preferred preservatives that may be
used in the pharmaceutical compositions of the present invention
include, but are not limited to, benzalkonium chloride,
chlorobutanol, thimerosal, phenylmercuric acetate and
phenylmercuric nitrate. A preferred surfactant is, for example,
Tween 80. Likewise, various preferred vehicles may be used in the
ophthalmic preparations of the present invention. These vehicles
include, but are not limited to, polyvinyl alcohol, povidone,
hydroxypropyl methyl cellulose, poloxamers, carboxymethyl
cellulose, hydroxyethyl cellulose cyclodextrin and purified
water.
[0137] Tonicity adjustors may be added as needed or convenient.
They include, but are not limited to, salts, particularly sodium
chloride, potassium chloride, mannitol and glycerin, or any other
suitable ophthalmically acceptable tonicity adjustor.
[0138] Various buffers and means for adjusting pH may be used so
long as the resulting preparation is ophthalmically acceptable.
Accordingly, buffers include acetate buffers, citrate buffers,
phosphate buffers and borate buffers. Acids or bases may be used to
adjust the pH of these formulations as needed.
[0139] In a similar manner an ophthalmically acceptable antioxidant
for use in the present invention includes, but is not limited to,
sodium metabisulfite, sodium thiosulfate, acetylcysteine, butylated
hydroxyanisole and butylated hydroxytoluene. Other excipient
components which may be included in the ophthalmic preparations are
chelating agents. The preferred chelating agent is edentate
disodium, although other chelating agents may also be used in place
of or in conjunction with it.
[0140] The ingredients are usually used in the following
amounts:
TABLE-US-00001 Ingredient Amount (% w/v) active ingredient about
0.001-5 preservative .sup. 0-0.10 vehicle 0-40 tonicity adjustor
1-10 buffer 0.01-10 .sup. pH adjustor q.s. pH 4.5-7.8 antioxidant
as needed surfactant as needed purified water to make 100%
[0141] The actual dose of the active compounds of the present
invention depends on the specific compound, and on the condition to
be treated; the selection of the appropriate dose is well within
the knowledge of the skilled artisan.
[0142] The ophthalmic formulations of the present invention are
conveniently packaged in forms suitable for metered application,
such as in containers equipped with a dropper, to facilitate
application to the eye. Containers suitable for dropwise
application are usually made of suitable inert, non-toxic plastic
material, and generally contain between about 0.5 and about 15 ml
solution. One package may contain one or more unit doses.
Especially preservative-free solutions are often formulated in
non-resealable containers containing up to about ten, preferably up
to about five units doses, where a typical unit dose is from one to
about 8 drops, preferably one to about 3 drops. The volume of one
drop usually is about 20-35 .mu.l.
[0143] The pharmaceutical compositions may be in the form of a
sterile injectable suspension. This suspension may be formulated
according to known methods using suitable dispersing or wetting
agents and suspending agents. The sterile injectable preparation
may also be a sterile injectable solution or suspension in a
non-toxic parenterally-acceptable diluent or solvent, for example,
as a solution in 1,3-butanediol. Sterile, fixed oils are
conventionally employed as a solvent or suspending medium. For this
purpose any bland fixed oil may be employed including synthetic
mono- or diglycerides, fatty acids (including oleic acid),
naturally occurring vegetable oils like sesame oil, coconut oil,
peanut oil, cottonseed oil, etc., or synthetic fatty vehicles like
ethyl oleate or the like. Buffers, preservatives, antioxidants, and
the like can be incorporated as required.
[0144] The compounds of the invention may also be administered in
the form of suppositories for rectal administration of the drug.
These compositions may be prepared by mixing the invention
compounds with a suitable non-irritating excipient, such as cocoa
butter, synthetic glyceride esters of polyethylene glycols, which
are solid at ordinary temperatures, but liquefy and/or dissolve in
the rectal cavity to release the drug.
[0145] Since individual subjects may present a wide variation in
severity of symptoms and each drug has its unique therapeutic
characteristics, the precise mode of administration and dosage
employed for each subject is left to the discretion of the
practitioner. The present invention is further directed to
pharmaceutical compositions comprising a pharmaceutically effective
amount of one or more of the above-described compounds and a
pharmaceutically acceptable carrier or excipient, wherein said
compositions are effective for treating the above diseases and
conditions; especially ophthalmic diseases and conditions. Such a
composition is believed to modulate signal transduction by a
tyrosine kinase, either by inhibition of catalytic activity,
affinity to ATP or ability to interact with a substrate.
More particularly, the compositions of the present invention may be
included in methods for treating diseases comprising proliferation,
fibrotic or metabolic disorders, for example cancer, fibrosis,
psoriasis, rosacea, atherosclerosis, arthritis, and other disorders
related to abnormal vasculogenesis and/or angiogenesis, such as
exudative age related macular degeneration and diabetic
retinopathy.
[0146] The present invention is further directed to pharmaceutical
compositions comprising a pharmaceutically effective amount of the
above-described compounds and a pharmaceutically acceptable carrier
or excipient. Such a composition is believed to modulate signal
transduction by a protein kinase, tyrosine kinase, either by
inhibition of catalytic activity, affinity to ATP or ability to
interact with a substrate.
[0147] The present invention relates to compounds capable of
regulating and/or modulating tyrosine kinase signal transduction
and more particularly receptor and non-receptor tyrosine kinase
signal transduction.
[0148] For preparing pharmaceutical compositions from the compounds
described by this invention, inert, pharmaceutically acceptable
carriers can be either solid or liquid. Solid form preparations
include powders, tablets, dispersible granules, capsules, cachets
and suppositories. The powders and tablets may be comprised of from
about 5 to about 95 percent active ingredient. Suitable solid
carriers are known in the art, e.g., magnesium carbonate, magnesium
stearate, talc, sugar or lactose. Tablets, powders, cachets and
capsules can be used as solid dosage forms suitable for oral
administration. Examples of pharmaceutically acceptable carriers
and methods of manufacture for various compositions may be found in
A. Gennaro (ed.), Remington's Pharmaceutical Sciences, 18.sup.th
Edition, (1990), Mack Publishing Co., Easton, Pa.
[0149] Liquid form preparations include solutions, suspensions and
emulsions. As an example may be mentioned water or water-propylene
glycol solutions for parenteral injection or addition of sweeteners
and opacifiers for oral solutions, suspensions and emulsions.
Liquid form preparations may also include solutions for intranasal
administration.
[0150] Aerosol preparations suitable for inhalation may include
solutions and solids in powder form, which may be in combination
with a pharmaceutically acceptable carrier, such as an inert
compressed gas, e.g. nitrogen.
[0151] Also included are solid form preparations that are intended
to be converted, shortly before use, to liquid form preparations
for either oral or parenteral administration. Such liquid forms
include solutions, suspensions and emulsions.
[0152] The compounds of the invention may also be deliverable
transdermally. The transdermal compositions can take the form of
creams, lotions, aerosols and/or emulsions and can be included in a
transdermal patch of the matrix or reservoir type as are
conventional in the art for this purpose.
[0153] The compounds of this invention may also be delivered
orally, subcutaneously, intravenously, intrathecally or some
suitable combination(s) thereof.
[0154] In addition to the common dosage forms set out above, the
compounds of this invention may also be administered by controlled
release means and/or delivery devices such as those described in
U.S. Pat. Nos. 3,845,770; 3,916,899; 3,536,809; 3,598,123;
3,630,200; 4,008,719; and 5,366,738.
[0155] For use where a composition for intravenous administration
is employed, a suitable daily dosage range for anti-inflammatory,
anti-atherosclerotic or anti-allergic use is from about 0.001 mg to
about 25 mg (preferably from 0.01 mg to about 1 mg) of a compound
of this invention per kg of body weight per day and for
cytoprotective use from about 0.1 mg to about 100 mg (preferably
from about 1 mg to about 100 mg and more preferably from about 1 mg
to about 10 mg) of a compound of this invention per kg of body
weight per day. For the treatment of diseases of the eye,
ophthalmic preparations for ocular administration comprising
0.001-1% by weight solutions or suspensions of the compounds of
this invention in an acceptable ophthalmic formulation may be
used.
[0156] Preferably, the pharmaceutical preparation is in a unit
dosage form. In such form, the preparation is subdivided into
suitably sized unit doses containing appropriate quantities of the
active component, e.g., an effective amount to achieve the desired
purpose.
[0157] The magnitude of prophylactic or therapeutic dose of a
compound of this invention will, of course, vary with the nature of
the severity of the condition to be treated and with the particular
compound and its route of administration. It will also vary
according to the age, weight and response of the individual
patient. It is understood that a specific daily dosage amount can
simultaneously be both a therapeutically effective amount, e.g.,
for treatment to slow progression of an existing condition, and a
prophylactically effective amount, e.g., for prevention of
condition.
[0158] The quantity of active compound in a unit dose of
preparation may be varied or adjusted from about 0.001 mg to about
500 mg. In one embodiment, the quantity of active compound in a
unit dose of preparation is from about 0.01 mg to about 250 mg. In
another embodiment, the quantity of active compound in a unit dose
of preparation is from about 0.1 mg to about 100 mg. In another
embodiment, the quantity of active compound in a unit dose of
preparation is from about 1.0 mg to about 100 mg. In another
embodiment, the quantity of active compound in a unit dose of
preparation is from about 1.0 mg to about 50 mg. In still another
embodiment, the quantity of active compound in a unit dose of
preparation is from about 1.0 mg to about 25 mg.
[0159] The actual dosage employed may be varied depending upon the
requirements of the patient and the severity of the condition being
treated. Determination of the proper dosage regimen for a
particular situation is within the skill of the art. For
convenience, the total daily dosage may be divided and administered
in portions during the day as required.
[0160] The amount and frequency of administration of the compounds
of the invention and/or the pharmaceutically acceptable salts
thereof will be regulated according to the judgment of the
attending clinician considering such factors as age, condition and
size of the patient as well as severity of the symptoms being
treated. A typical recommended daily dosage regimen for oral
administration can range from about 0.01 mg/day to about 2000
mg/day of the compounds of the present invention. In one
embodiment, a daily dosage regimen for oral administration is from
about 1 mg/day to 1000 mg/day. In another embodiment, a daily
dosage regimen for oral administration is from about 1 mg/day to
500 mg/day. In another embodiment, a daily dosage regimen for oral
administration is from about 100 mg/day to 500 mg/day. In another
embodiment, a daily dosage regimen for oral administration is from
about 1 mg/day to 250 mg/day. In another embodiment, a daily dosage
regimen for oral administration is from about 100 mg/day to 250
mg/day. In still another embodiment, a daily dosage regimen for
oral administration is from about 1 mg/day to 100 mg/day. In still
another embodiment, a daily dosage regimen for oral administration
is from about 50 mg/day to 100 mg/day. In a further embodiment, a
daily dosage regimen for oral administration is from about 1 mg/day
to 50 mg/day. In another embodiment, a daily dosage regimen for
oral administration is from about 25 mg/day to 50 mg/day. In a
further embodiment, a daily dosage regimen for oral administration
is from about 1 mg/day to 25 mg/day. The daily dosage may be
administered in a single dosage or can be divided into from two to
four divided doses.
[0161] In one aspect, the present invention provides a kit
comprising a therapeutically effective amount of at least one
compound of the present invention, or a pharmaceutically acceptable
salt of said compound and a pharmaceutically acceptable carrier,
vehicle or diluents, and directions for the use of said kit.
[0162] The present invention is not to be limited in scope by the
specific embodiments disclosed in the examples which are intended
as illustrations of a few aspects of the invention and any
embodiments that are functionally equivalent are within the scope
of this invention. Indeed, various modifications of the invention
in addition to those shown and described herein will become
apparent to those skilled in the relevant art and are intended to
fall within the scope of the appended claims.
[0163] Receptor tyrosine kinase mediated signal transduction is
initiated by extracellular interaction with a specific growth
factor (ligand), followed by receptor dimerization, transient
stimulation of the intrinsic protein tyrosine kinase activity and
phosphorylation. Binding sites are thereby created for
intracellular signal transduction molecules and lead to the
formation of complexes with a spectrum of cytoplasmic signaling
molecules that facilitate the appropriate cellular response (e.g.,
cell division, metabolic effects and responses to the extracellular
microenvironment).
[0164] It has been shown that tyrosine phosphorylation sites in
growth factor receptors function as high-affinity binding sites for
SH2 (src homology) domains of signaling molecules. Several
intracellular substrate proteins that associate with receptor
tyrosine kinases have been identified. They may be divided into two
principal groups: (1) substrates which have a catalytic domain; and
(2) substrates which lack such domain but serve as adapters and
associate with catalytically active molecules. The specificity of
the interactions between receptors and SH2 domains of their
substrates is determined by the amino acid residues immediately
surrounding the phosphorylated tyrosine residue. Differences in the
binding affinities between SH2 domains and the amino acid sequences
surrounding the phosphotyrosine residues on particular receptors
are consistent with the observed differences in their substrate
phosphorylation profiles. These observations suggest that the
function of each receptor tyrosine kinase is determined not only by
its pattern of expression and ligand availability but also by the
array of downstream signal transduction pathways that are activated
by a particular receptor. Thus, phosphorylation provides an
important regulatory step which determines the selectivity of
signaling pathways recruited by specific growth factor receptors,
as well as differentiation factor receptors.
[0165] Tyrosine kinase signal transduction results in, among other
responses, cell proliferation, differentiation and metabolism.
Abnormal cell proliferation may result in a wide array of disorders
and diseases, including the development of neoplasia such as
carcinoma, sarcoma, leukemia, glioblastoma, hemangioma, psoriasis,
arteriosclerosis, arthritis and diabetic retinopathy (or other
disorders related to uncontrolled angiogenesis and/or
vasculogenesis, e.g. macular degeneration).
[0166] This invention is therefore directed to compounds which
regulate, modulate and/or inhibit tyrosine kinase signal
transduction by affecting the enzymatic activity of the RTKs and/or
the non-receptor tyrosine kinases and interfering with the signal
transduced by such proteins. More particularly, the present
invention is directed to compounds which regulate, modulate and/or
inhibit the RTK and/or non-receptor tyrosine kinase mediated signal
transduction pathways as a therapeutic approach to cure many kinds
of solid tumors, including but not limited to carcinoma, sarcoma,
leukemia, erythroblastoma, glioblastoma, meningioma, astrocytoma,
melanoma and myoblastoma. Indications may include, but are not
limited to brain cancers, bladder cancers, ovarian cancers, gastric
cancers, pancreas cancers, colon cancers, blood cancers, lung
cancers and bone cancers.
[0167] The present invention concerns also processes for preparing
the compounds of Formula I. The compounds of formula I according to
the invention can be prepared analogously to conventional methods
as understood by the person skilled in the art of synthetic organic
chemistry. Synthetic Schemes set forth below, illustrate how the
compounds according to the invention can be made.
##STR00019##
##STR00020##
##STR00021##
##STR00022##
##STR00023##
[0168] At this stage, those skilled in the art will appreciate that
many additional compounds that fall under the scope of the
invention may be prepared by performing various common chemical
reactions. Details of certain specific chemical transformations are
provided in the examples.
[0169] Those skilled in the art will be able to routinely modify
and/or adapt the following scheme to synthesize any compounds of
the invention covered by Formula I.
The present invention is not to be limited in scope by the
exemplified embodiments which are intended as illustrations of
single aspects of the invention only. Indeed, various modifications
of the invention in addition to those described herein will become
apparent to those skilled in the art from the foregoing
description.
DETAILED DESCRIPTION OF THE INVENTION
[0170] The present invention relates to organic molecules capable
of modulating, regulating and/or inhibiting protein kinase signal
transduction, useful for treating diseases related to protein
kinase signal transduction, for example, cancer, blood vessel
proliferative disorders, fibrotic disorders, and neurodegenerative
diseases. In particular, the compounds of the present invention are
useful for treatment of mesangial cell proliferative disorders and
metabolic diseases, lung carcinomas, breast carcinomas, Non
Hodgkin's lymphomas, ovarian carcinoma, pancreatic cancer,
malignant pleural mesothelioma, melanoma, arthritis, restenosis,
hepatic cirrhosis, atherosclerosis, psoriasis, rosacea, diabetic
mellitus, wound healing, inflammation and neurodegenerative
diseases and preferably ophthalmic diseases, i.e. diabetic
retinopathy, retinopathy of prematurity, macular edema, retinal
vein occlusion, exudative or neovascular age-related macular
degeneration, high-risk eyes (i.e. fellow eyes have neovascular
age-related macular degeneration) with dry age-related macular
degeneration, neovascular disease associated with retinal vein
occlusion, neovascular disease (including choroidal
neovascularization) associated with the following: pathologic
myopia, pseudoxanthoma elasticum, optic nerve drusen, traumatic
choroidal rupture, atrophic macular degeneration, geographic
atrophy, central serous retinopathy, cystoid macular edema,
diabetic retinopathy, proliferative diabetic retinopathy, diabetic
macular edema, rubeosis iridis, retinopathy of prematurity, Central
and branch retinal vein occlusions, inflammatory/infectious
retinal, neovascularization/edema, corneal neovascularization,
hyperemia related to an actively inflamed pterygia, recurrent
pterygia following excisional surgery, post-excision, progressive
pterygia approaching the visual axis, prophylactic therapy to
prevent recurrent pterygia, of post-excision, progressive pterygia
approaching the visual axis, chronic low grade hyperemia associated
with pterygia, neovascular glaucoma, iris neovascularization,
idiopathic etiologies, presumed ocular histoplasmosis syndrome,
retinopathy of prematurity, chronic allergic conjunctivitis, ocular
rosacea, blepharoconjunctivitis, recurrent episcleritis,
keratoconjunctivitis sicca, ocular graft vs host disease, etc.
[0171] In another aspect, the invention provides the use of at
least one kinase inhibitor for the manufacture of a medicament for
the treatment of a disease or a condition mediated by tyrosine
kinases in a mammal.
[0172] It is to be understood that both the foregoing general
description and the following detailed description are exemplary
and explanatory only and are not restrictive of the invention
claimed. As used herein, the use of the singular includes the
plural unless specifically stated otherwise.
[0173] It will be readily apparent to those skilled in the art that
some of the compounds of the invention may contain one or more
asymmetric centers, such that the compounds may exist in
enantiomeric as well as in diastereomeric forms. Unless it is
specifically noted otherwise, the scope of the present invention
includes all enantiomers, diastereomers and racemic mixtures. Some
of the compounds of the invention may form salts with
pharmaceutically acceptable acids or bases, and such
pharmaceutically acceptable salts of the compounds described herein
are also within the scope of the invention.
[0174] The present invention includes all pharmaceutically
acceptable isotopically enriched compounds. Any compound of the
invention may contain one or more isotopic atoms enriched or
different than the natural ratio such as deuterium .sup.2H (or D)
in place of hydrogen .sup.1H (or H) or use of .sup.13C enriched
material in place of .sup.12C and the like. Similar substitutions
can be employed for N, O and S. The use of isotopes may assist in
analytical as well as therapeutic aspects of the invention. For
example, use of deuterium may increase the in vivo half-life by
altering the metabolism (rate) of the compounds of the invention.
These compounds can be prepared in accord with the preparations
described by use of isotopically enriched reagents.
[0175] The following examples are for illustrative purposes only
and are not intended, nor should they be construed as limiting the
invention in any manner. Those skilled in the art will appreciate
that variations and modifications of the following examples can be
made without exceeding the spirit or scope of the invention.
[0176] As will be evident to those skilled in the art, individual
isomeric forms can be obtained by separation of mixtures thereof in
conventional manner. For example, in the case of diastereoisomeric
isomers, chromatographic separation may be employed.
[0177] Compound names were generated with ACDLabs version 12.5.
Some of the intermediate and reagent names used in the examples
were generated with software such as Chem Bio Draw Ultra version
12.0 or Auto Nom 2000 from MDL ISIS Draw 2.5 SP1.
[0178] In general, characterization of the compounds is performed
according to the following methods; NMR spectra are recorded on 300
or 600 MHz Varian and acquired at room temperature. Chemical shifts
are given in ppm referenced either to internal TMS or to the
solvent signal.
[0179] All the reagents, solvents, catalysts for which the
synthesis is not described are purchased from chemical vendors such
as Sigma Aldrich, Fluka, Bio-Blocks, Combi-blocks, TCI, VWR,
Lancaster, Oakwood, Trans World Chemical, Alfa, Fisher, Maybridge,
Frontier, Matrix, Ukrorgsynth, Toronto, Ryan Scientific, SiliCycle,
Anaspec, Syn Chem, Chem-Impex, MIC-scientific, Ltd; however some
known intermediates, were prepared according to published
procedures.
[0180] Usually the compounds of the invention were purified by
medium pressure liquid chromatography, unless noted otherwise.
Examples
##STR00024##
[0181]
3-(5-amino-1-benzothien-2-yl)-5-(2H-tetrazol-5-yl)pyridin-2-amine
[0182] The reaction mixture of
3-iodo-5-(2H-tetrazol-5-yl)pyridin-2-amine (288 mg, 1.0 mmol, 1
eq),
2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzo[b]thiophen-5-amine
(330 mg, 1.2 eq), triphenylphosphine (53 mg, 0.2 eq), and palladium
diacetate (22 mg, 0.1 eq) in dioxane (3 mL) and aqueous sodium
carbonate (2 M, 2 mL, 4 eq) under nitrogen atmosphere was
vigorously stirred and heated at 40.degree. C. for 30 minutes. The
mixture was then partitioned between aqueous ammonium chloride and
THF-EtOAc (1:10). The organic layer was isolated and dried with
anhydrous sodium sulfate. The upper solution was concentrated down
with silica gel. Upon a gradient column chromatography (EtOAc to
MeOH-EtOAc 1:10)
3-(5-amino-1-benzothien-2-yl)-5-(2H-tetrazol-5-yl)pyridin-2-amine
was obtained in the amount of 5 mg.
[0183] .sup.1H NMR (600 MHz, DMSO-d.sub.6) .delta. ppm 8.57 (d,
J=1.76 Hz, 1H) 8.07 (d, J=1.76 Hz, 1H) 7.59 (d, J=8.51 Hz, 1H) 7.43
(s, 1H) 6.98 (d, J=1.76 Hz, 1H) 6.73 (dd, J=8.51, 1.91 Hz, 1H) 6.06
(br. s., 2H) 5.11 (br. s. 2H)
##STR00025##
1-[4-chloro-3-(trifluoromethyl)phenyl]-3-{2-[5-(2H-tetrazol-5-yl)pyridin--
3-yl]-1-benzothien-5-yl}urea
[0184] To the solution of 5-bromo-3-cyanopyridine (3.0 g, 15.9
mmol, 1 eq) in anhydrous dimethylformamide (40 mL) was added
ammonium chloride (1.45 g, 1.7 eq) and sodium azide (1.76 g, 1.7
eq). The resulting reaction mixture was heated at 100.degree. C.
under nitrogen for 20 hours. After the reaction mixture was cooled
to room temperature, it was poured into ice-water and the pH of the
mixture was adjusted to .about.3.5 using aqueous hydrochloric acid
(2 N). The aqueous was first extracted with ethyl acetate three
times, followed by an extraction with i-PrOH--CHCl.sub.3 (1:4). All
organics were combined and dried with anhydrous sodium sulfate. The
upper clear liquor was decanted, concentrated under reduced
pressure, and the resulting oily residue was placed under high
vacuum for 20 hours. Upon treatment with EtOAc-Hex (1:1) and
filtration, 3-bromo-5-(2H-tetrazol-5-yl)pyridine was obtained as a
white solid in the amount of 3.241 g.
[0185] .sup.1H NMR (600 MHz, DMSO-d.sub.6) .delta. ppm 9.19 (d,
J=1.76 Hz, 1H) 8.92 (d, J=2.20 Hz, 1H) 8.61 (t, J=2.05 Hz, 1H)
[0186] The reaction mixture of 3-bromo-5-(2H-tetrazol-5-yl)pyridine
(226 mg, 1.0 mmol, 1 eq),
2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzo[b]thiophen-5-amine
(316 mg, 1.15 eq), triphenylphosphine (53 mg, 0.2 eq), and
palladium diacetate (22 mg, 0.1 eq) in dioxane (3 mL) and aqueous
sodium carbonate (2 M, 2 mL, 4 eq) under nitrogen atmosphere was
vigorously stirred and heated at 100.degree. C. for three hours. It
was then cooled to room temperature, filtered through a Buchner
funnel, and rinsed with small amount of water and ethyl acetate.
The filtrate was isolated and concentrated down with silica gel.
Upon a gradient column chromatography (EtOAc to MeOH-EtOAc 1:4),
2-(5-(2H-tetrazol-5-yl)pyridin-3-yl)benzo[b]thiophen-5-amine was
obtained as an orange colored solid in the amount of 92 mg while
the starting material, 3-bromo-5-(2H-tetrazol-5-yl)pyridine was
recovered in the amount of 90 mg.
[0187] .sup.1H NMR (300 MHz, DMSO-d.sub.6) .delta. ppm 9.08 (s, 1H)
8.87 (d, J=1.76 Hz, 1H) 8.48 (s, 1H) 7.80 (s, 1H) 7.62 (d, J=8.50
Hz, 1H) 7.01 (d, J=0.73 Hz, 1H) 6.75 (dd, J=7.98, 0.95 Hz, 1H) 5.16
(br. s., 2H)
[0188] The reaction mixture of
2-(5-(2H-tetrazol-5-yl)pyridin-3-yl)benzo[b]thiophen-5-amine (44.1
mg, 0.15 mmol, 1 eq) and 4-chloro-3-(trifluoromethyl)phenyl
isocyanate (33.2 mg, 1 eq) in anhydrous tetrahydrofuran (1.5 mL)
under nitrogen atmosphere was stirred at room temperature for 45
minutes. The reaction was then diluted with ethyl acetate, washed
with aqueous ammonium chloride, brine, and dried with anhydrous
sodium sulfate. The upper clear solution was decanted and
concentrated. The soft solid was wrapped with silica gel and
chromatographed (EtOAc to MeOH-EtOAc 1:4) to give
1-[4-chloro-3-(trifluoromethyl)phenyl]-3-{2-[5-(2H-tetrazol-5-yl)pyridin--
3-yl]-1-benzothien-5-yl}urea as a yellow powder in the amount of 26
mg.
[0189] .sup.1H NMR (600 MHz, DMSO-d.sub.6) .delta. ppm 9.42 (br.
s., 1H) 9.20 (br. s., 1H) 9.12 (d, J=1.91 Hz, 1H) 8.93 (d, J=2.35
Hz, 1H) 8.53 (t, J=2.13 Hz, 1H) 8.16 (d, J=2.49 Hz, 1H) 8.13 (d,
J=2.05 Hz, 1H) 8.04 (s, 1H) 7.94 (d, J=8.66 Hz, 1H) 7.68 (dd,
J=8.73, 2.42 Hz, 1H) 7.61-7.64 (m, 1H) 7.44 (dd, J=8.80, 2.05 Hz,
1H).
##STR00026##
1-{2-[2-amino-5-(2H-tetrazol-5-yl)pyridin-3-yl]-1-benzothien-5-yl}-3-[4-c-
hloro-3-(trifluoromethyl)phenyl]urea
[0190] To the stirring mixture of
3-(5-aminobenzo[b]thiophen-2-yl)-5-isocyanopyridin-2-amine (1.064
g, 4 mmol, 1 eq) in anhydrous tetrahydrofuran (25 mL) under
nitrogen atmosphere was added 4-chloro-3-(trifluoromethyl)phenyl
isocyanate (904 mg, 1 eq) and the reaction mixture was stirred at
room temperature for three hours. The reaction was then diluted
with ethyl acetate, washed sequentially with aqueous ammonium
chloride, saturated aqueous sodium bicarbonate, and brine, and
dried with anhydrous sodium sulfate. The upper clear solution was
decanted and concentrated under reduced pressure to lesser amount,
to which was added a small amount of EtOAc-Hex (2:1). Upon a
filtration,
1-(2-(2-amino-5-isocyanopyridin-3-yl)benzo[b]thiophen-5-yl)-3-(4-chloro-3-
-(trifluoromethyl)phenyl)urea was obtained as white solid in the
amount of 1.37 g.
[0191] .sup.1H NMR (600 MHz, DMSO-d.sub.6) .delta. ppm 9.23 (s, 1H)
9.03 (s, 1H) 8.42 (d, J=2.05 Hz, 1H) 8.16 (d, J=2.05 Hz, 1H) 8.13
(d, J=1.76 Hz, 1H) 7.89-7.92 (m, 2H) 7.64-7.67 (m, 1H) 7.60-7.64
(m, 2H) 7.40 (dd, J=8.80, 1.76 Hz, 1H) 7.15 (br. s., 2H).
[0192] The mixture of
1-(2-(2-amino-5-isocyanopyridin-3-yl)benzo[b]thiophen-5-yl)-3-(4-chloro-3-
-(trifluoromethyl)phenyl)urea (1.3 g, 2.67 mmol, 1 eq), ammonium
chloride (286 mg, 2 eq), and sodium azide (347 mg, 2 eq) in
anhydrous dimethylformamide (8 mL) under nitrogen atmosphere was
stirred and heated at 120.degree. C. for 2 hours. The mixture was
then cooled to room temperature and additional ammonium chloride
(286 mg, 2 eq) and sodium azide (347 mg, 2 eq) were added. The
reaction mixture was heated back to 120.degree. C. and stirred at
that temperature for another hour. After it was cooled to room
temperature, it was transferred into a mixture of ethyl acetate and
aqueous ammonium chloride; during the process, the pH of the
aqueous layer was adjusted to 3.about.4ish by using aq KHSO.sub.4
(10%). The organic layer was isolated, washed once with saturated
brine, and dried with anhydrous sodium sulfate. The upper solution
was decanted and concentrated down with silica gel. Upon gradient
column chromatography (from EtOAc to MeOH-EtOAc 1:2),
1-{2-[2-amino-5-(2H-tetrazol-5-yl)pyridin-3-yl]-1-benzothien-5-yl}-3-[4-c-
hloro-3-(trifluoromethyl)phenyl]urea was obtained as a yellow solid
in the amount of 452 mg.
[0193] .sup.1H NMR (600 MHz, DMSO-d.sub.6) .delta. ppm 9.29 (s, 1H)
9.07 (s, 1H) 8.61 (s, 1H) 8.16 (br. s., 1H) 8.11 (br. s., 2H) 7.91
(d, J=8.51 Hz, 1H) 7.65-7.69 (m, 2H) 7.61-7.64 (m, 1H) 7.41 (d,
J=8.22 Hz, 1H) 6.28 (br. s., 2H).
##STR00027##
5-(2-(3-((tert-butyldimethylsilyl)oxy)propyl)-2H-tetrazol-5-yl)-3-iodopyr-
idin-2-amine
[0194] The mixture of 6-amino-5-bromonicotinonitrile (3.315 g, 15.9
mmol, 1 eq), sodium iodide (4.77 g, 2 eq), copper(I) iodide (303
mg, 0.1 eq), and trans-N,N'-dimethylcyclohexane-1,2-diamine (0.52
mL, 0.2 eq) in anhydrous dioxane (40 mL under nitrogen atmosphere
was stirred at 120.degree. C. for 20 hours. The mixture was cooled
to room temperature then partitioned between ethyl acetate and
aqueous ammonium chloride. The organic layer was isolated, further
washed with saturated aqueous sodium bicarbonate, brine, and dried
with anhydrous sodium sulfate. The upper solution was decanted,
concentrated, and the crude solid residue was subject to a column
chromatography started first with EtOAc-Hex (1:5 to 1:1) followed
by MeOH--CHCl3 (1:100 to 1:20). Product containing fractions were
all collected and concentrated. The solid residue was triturated
with EtOAc-Hex (1:4) yielding 3-iodo-5-isocyanopyridin-2-amine as
an off-white solid in the amount of 2.75 g upon filtration.
[0195] .sup.1H NMR (600 MHz, DMSO-d.sub.6) .delta. ppm 8.35 (d,
J=1.76 Hz, 1H) 8.30 (d, J=1.76 Hz, 1H) 7.14 (br. s., 2H).
[0196] To the solution of 3-iodo-5-isocyanopyridin-2-amine (2.74 g,
11.2 mmol, 1 eq) in anhydrous dimethylformamide (25 mL) in a 100 mL
round bottom flask was added ammonium chloride (1.02 g, 1.7 eq) and
sodium azide (1.24 g, 1.7 eq). The resulting reaction mixture was
heated to 100.degree. C. under nitrogen for 20 hours. After the
reaction mixture was cooled to room temperature, it was poured into
ice-chunk filled water and the pH of the mixture was adjusted to
.about.3.5 using aqueous hydrochloric acid (2 N). After the mixture
was stirred at room temperature for about two hours, it was
filtered through a Buchner funnel, rinsed with water, giving a
brown solid. The solid was treated with MeOH--CHCl.sub.3 and
concentrated down with silica gel. Upon gradient column
chromatography (MeOH-EtOAc 1:20 to 1:5),
3-iodo-5-(2H-tetrazol-5-yl)pyridin-2-amine was obtained as
off-white solid in the amount of 1.15 g.
[0197] .sup.1H NMR (600 MHz, DMSO-d.sub.6) .delta. ppm 8.60 (d,
J=1.76 Hz, 1H) 8.45 (d, J=2.05 Hz, 1H) 6.77 (br. s., 2H).
[0198] The reaction mixture of
3-iodo-5-(2H-tetrazol-5-yl)pyridin-2-amine (0.61 g, 2.12 mmol, 1
eq), (3-bromopropoxy)-tert-butyldimethylsilane (0.66 mL, 1.3 eq),
and potassium carbonate (0.44 g, 1.5 eq) in anhydrous
dimethylformamide (8 mL) was stirred and heated at 80.degree. C.
under nitrogen atmosphere for 2 hours. It was then diluted with
ethyl acetate, washed sequentially with aqueous ammonium chloride,
saturated aqueous sodium bicarbonate, and brine, and dried with
anhydrous sodium sulfate. The upper clear solution was decanted and
concentrated under reduced pressure with silica gel. A gradient
column chromatography [Hexane to EtOAc-Hex (1:2)] rendered
5-(2-(3-((tert-butyldimethylsilyl)oxy)propyl)-2H-tetrazol-5-yl)-3-iodopyr-
idin-2-amine as a white solid in the amount of 797 mg.
[0199] .sup.1H NMR (600 MHz, DMSO-d.sub.6) .delta. ppm 8.60 (d,
J=1.76 Hz, 1H) 8.40 (d, J=1.76 Hz, 1H) 6.63 (br. s., 2H) 4.74 (t,
J=6.75 Hz, 2H) 3.63 (t, J=5.72 Hz, 2H) 2.14 (quin, J=6.24 Hz, 2H)
0.85 (s, 9H) 0.01 (s, 6H).
##STR00028##
1-(2-{2-amino-5-[2-(3-hydroxypropyl)-2H-tetrazol-5-yl]
pyridin-3-yl}-1-benzothien-5-yl)-3-[4-chloro-3-(trifluoromethyl)phenyl]ur-
ea
[0200] To the solution of tert-butyl
(2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzo[b]thiophen-5-yl)car-
bamate (7.51 g, 20 mmol, 1 eq) in anhydrous dichloromethane (40 mL
under nitrogen atmosphere at 0.degree. C. was added dropwise
trifluoroacetic acid (15.4 mL, 10 eq) and the reaction was stirred
at 0.degree. C. for 30 minutes followed by at room temperature for
about two hours. The reaction was then slowly poured into an
ice-cold saturated aqueous sodium bicarbonate solution with
stirring and the white solid that appeared during the process was
filtered, washed with water, to give a first batch of
2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzo[b]thiophen-5-amin-
e in the amount of 2.792 g. The pH of the filtrate was adjusted to
8 by addition of solid sodium bicarbonate with stirring and then
extracted using chloroform (3.times.). All organic layers were
combined, dried with anhydrous sodium sulfate, and concentrated
down to give brown oil. After the oil was placed in vacuo for two
hours, it was treated with EtOAc-Hex (1:9) and the mixture was
stirred at room temperature for 30 minutes. A second batch of
2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzo[b]thiophen-5-amine
was isolated as a pale pink solid in the amount of 1.883 g upon
filtration. The total amount of
2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzo[b]thiophen-5-amine
is 4.675 g with a yield of 85%.
[0201] .sup.1H NMR (600 MHz, DMSO-d.sub.6) .delta. ppm 7.60-7.62
(m, 2H) 7.01 (d, J=2.05 Hz, 1H) 6.80 (dd, J=8.66, 2.20 Hz, 1H) 5.18
(br. s., 2H) 1.30 (s, 12H)
[0202] To the solution of
2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzo[b]thiophen-5-amine
(825 mg, 3 mmol, 1 eq) in anhydrous tetrahydrofuran (15 mL) was
added 4-chloro-3-(trifluoromethyl)phenyl isocyanate (678.3 mg, 1
eq) and the reaction solution was stirred at room temperature under
nitrogen atmosphere overnight. The reaction was then diluted with
ethyl acetate, washed sequentially with aqueous ammonium chloride,
saturated aqueous sodium bicarbonate, and brine, and dried with
anhydrous sodium sulfate. The upper clear solution was decanted and
concentrated under reduced pressure.
1-(4-chloro-3-(trifluoromethyl)phenyl)-3-(2-(4,4,5,5-tetramethy-
l-1,3,2-dioxaborolan-2-yl)benzo[b]thiophen-5-yl)urea was obtained
as slightly brown colored foam in vacuo in the amount of 1.5 g
which was used directly without further purification.
[0203] .sup.1H NMR (600 MHz, DMSO-d.sub.6) .delta. ppm 9.18 (s, 1H)
8.99 (s, 1H) 8.15 (dd, J=10.12, 1.91 Hz, 2H) 7.92 (d, J=8.80 Hz,
1H) 7.85 (s, 1H) 7.64-7.66 (m, 1H) 7.61-7.63 (m, 1H) 7.44 (dd,
J=8.80, 1.76 Hz, 1H) 1.33 (s, 12H).
[0204] To the mixture of
5-(2-(3-((tert-butyldimethylsilyl)oxy)propyl)-2H-tetrazol-5-yl)-3-iodopyr-
idin-2-amine (184 mg, 0.4 mmol, 1 eq) and
1-(4-chloro-3-(trifluoromethyl)phenyl)-3-(2-(4,4,5,5-tetramethyl-1,3,2-di-
oxaborolan-2-yl)benzo[b]thiophen-5-yl)urea (218 mg, 1.1 eq) in
dioxane (3 mL) and water (0.75 mL) under nitrogen atmosphere was
added potassium acetate (157 mg, 4 eq) and
[1,1'-bis(diphenylphosphino)ferrocene] dichloropalladium(III)
(complex with dichloromethane, 65 mg, 0.2 eq). After the mixture
was stirred at 40.degree. C. for 30 minutes, additional amount of
(46 mg) and palladium catalyst (50 mg) were added and the reaction
was continued at that temperature for an hour. The mixture was then
diluted with ethyl acetate, washed sequentially with aqueous
ammonium chloride, saturated aqueous sodium bicarbonate, and brine,
and dried with anhydrous sodium sulfate. The upper clear solution
was decanted and concentrated and loaded onto silica. Gradient
column chromatography (EtOAc-Hex 1:4 to 6:1) gave a brown solid
which was triturated with EtOAc-Hex (3:2) rendering
1-(2-(2-amino-5-(2-(3-((tert-butyldimethylsilyl)oxy)propyl)-2H-tetrazol-5-
-yl)pyridin-3-yl)benzo[b]thiophen-5-yl)-3-(4-chloro-3-(trifluoromethyl)phe-
nyl)urea as an off-white solid in the amount of 90 mg.
[0205] .sup.1H NMR (600 MHz, DMSO-d.sub.6) .delta. ppm 9.20 (s, 1H)
9.00 (s, 1H) 8.67 (d, J=2.05 Hz, 1H) 8.16 (d, J=2.05 Hz, 1H) 8.13
(d, J=1.76 Hz, 1H) 8.09 (d, J=2.05 Hz, 1H) 7.91 (d, J=8.51 Hz, 1H)
7.69 (s, 1H) 7.64-7.67 (m, 1H) 7.61-7.64 (m, 1H) 7.41 (dd, J=8.66,
1.91 Hz, 1H) 6.68 (s, 2H) 4.76 (t, J=6.75 Hz, 2H) 3.65 (t, J=5.72
Hz, 2H) 2.16 (quin, J=6.24 Hz, 2H) 0.85 (s, 9H) 0.02 (s, 6H).
[0206] To the solution of
1-(2-(2-amino-5-(2-(3-((tert-butyldimethylsilyl)oxy)propyl)-2H-tetrazol-5-
-yl)pyridin-3-yl)benzo[b]thiophen-5-yl)-3-(4-chloro-3-(trifluoromethyl)phe-
nyl)urea (100 mg, 0.142 mmol, 1 eq) in anhydrous tetrahydrofuran (3
mL) under nitrogen atmosphere at 0.degree. C. was added dropwise a
solution of tetrabutylammonium fluoride (1.0 M in THF, 0.43 mL, 3
eq). The reaction was stirred at room temperature for 2 hours and
then partitioned between ethyl acetate and aqueous ammonium
chloride. The organic layer was isolated, further washed with
saturated aqueous sodium bicarbonate, brine, and dried with
anhydrous sodium sulfate. The upper clear solution was decanted and
concentrated and loaded onto silica. The mixture was subject to a
gradient column chromatography (EtOAc-Hex 4:1 to MeOH-EtOAc 1:100).
The product fractions were collected and concentrated. The solid
residue was triturated in ethyl acetate yielding
1-(2-{2-amino-5-[2-(3-hydroxypropyl)-2H-tetrazol-5-yl]pyridin-3-yl}-1-ben-
zothien-5-yl)-3-[4-chloro-3-(trifluoromethyl)phenyl]urea as a white
solid in the amount of 50 mg.
[0207] .sup.1H NMR (600 MHz, DMSO-d.sub.6) .delta. ppm 9.21 (s, 1H)
9.01 (s, 1H) 8.68 (d, J=2.05 Hz, 1H) 8.16 (d, J=2.05 Hz, 1H) 8.13
(d, J=1.76 Hz, 1H) 8.09 (d, J=2.05 Hz, 1H) 7.91 (d, J=8.51 Hz, 1H)
7.69 (s, 1H) 7.64-7.67 (m, 1H) 7.61-7.64 (m, 1H) 7.41 (dd, J=8.66,
1.91 Hz, 1H) 6.68 (s, 2H) 4.76 (t, J=7.04 Hz, 2H) 4.70 (t, J=4.99
Hz, 1H) 3.47 (q, J=5.67 Hz, 2H) 2.11 (quin, J=6.60 Hz, 2H)
##STR00029##
1-(2-{2-amino-5-[2-(3-hydroxypropyl)-2H-tetrazol-5-yl]
pyridin-3-yl}-1-benzothien-5-yl)-3-[4-fluoro-3-(trifluoromethyl)phenyl]ur-
ea
[0208] Synthesized using a procedure similar to the one used for
the synthesis of Compound 3.
[0209] .sup.1H NMR (600 MHz, DMSO-d.sub.6) .delta. ppm 9.06 (s, 1H)
8.95 (s, 1H) 8.68 (s, 1H) 8.11 (d, J=17.61 Hz, 2H) 8.05 (d, J=4.40
Hz, 1H) 7.90 (d, J=8.80 Hz, 1H) 7.69 (s, 1H) 7.63-7.68 (m, 1H) 7.45
(t, J=9.68 Hz, 1H) 7.41 (d, J=8.51 Hz, 1H) 6.68 (br. s., 2H) 4.76
(t, J=6.90 Hz, 2H) 4.70 (t, J=4.84 Hz, 1H) 3.47 (q, J=5.58 Hz, 2H)
2.11 (quin, J=6.46 Hz, 2H).
##STR00030##
1-(2-{2-amino-5-[2-(3-hydroxypropyl)-2H-tetrazol-5-yl]
pyridin-3-yl}-1-benzothien-5-yl)-3-[3-fluoro-5-(trifluoromethyl)phenyl]ur-
ea
[0210] Synthesized using a procedure similar to the one used for
the synthesis of Compound 3.
[0211] .sup.1H NMR (600 MHz, DMSO-d.sub.6) .delta. ppm 9.30 (br.
s., 1H) 9.09 (br. s., 1H) 8.68 (d, J=0.88 Hz, 1H) 8.13 (s, 1H) 8.09
(s, 1H) 7.92 (d, J=8.66 Hz, 1H) 7.75 (s, 1H) 7.70 (s, 1H) 7.64 (d,
J=11.15 Hz, 1H) 7.42 (d, J=8.51 Hz, 1H) 7.23 (d, J=8.22 Hz, 1H)
6.68 (br. s., 2H) 4.76 (t, J=6.90 Hz, 2H) 4.70 (t, J=4.84 Hz, 1H)
3.47 (q, J=5.53 Hz, 2H) 2.11 (quin, J=6.35 Hz, 2H).
##STR00031##
1-(2-{2-amino-5-[2-(3-hydroxypropyl)-2H-tetrazol-5-yl]
pyridin-3-yl}-1-benzothien-5-yl)-3-[3,5-bis(trifluoromethyl)phenyl]urea
[0212] Synthesized using a procedure similar to the one used for
the synthesis of Compound 3.
[0213] .sup.1H NMR (600 MHz, DMSO-d.sub.6) .delta. ppm 9.46 (br.
s., 1H) 9.18 (br. s., 1H) 8.68 (d, J=1.47 Hz, 1H) 8.17 (s, 2H) 8.15
(s, 1H) 8.10 (d, J=1.17 Hz, 1H) 7.92 (d, J=8.80 Hz, 1H) 7.70 (s,
1H) 7.65 (s, 1H) 7.44 (d, J=8.51 Hz, 1H) 6.68 (br. s., 2H) 4.76 (t,
J=6.90 Hz, 2H) 4.70 (t, J=4.84 Hz, 1H) 3.47 (q, J=5.77 Hz, 2H) 2.11
(quin, J=6.46 Hz, 2H)
##STR00032##
1-(2-{2-amino-5-[1-(3-hydroxypropyl)-1H-tetrazol-5-yl]pyridin-3-yl}-1-ben-
zothien-5-yl)-3-[4-chloro-3-(trifluoromethyl)phenyl]urea
[0214] To the solution of
5-(1-(3-((tert-butyldimethylsilyl)oxy)propyl)-1H-tetrazol-5-yl)-3-iodopyr-
idin-2-amine (184 mg, 0.4 mmol, 1 eq),
2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzo[b]thiophen-5-amine
(165 mg, 1.5 eq), and triphenylphosphine (21 mg, 0.2 eq) in dioxane
(2 mL) and aqueous sodium carbonate (2M, 0.8 mL, 4 eq) under
nitrogen atmosphere, was added palladium diacetate (9 mg, 0.1 eq)
and the reaction mixture was vigorously stirred at 40.degree. C.
for two hours. It was then partitioned between ethyl acetate and
aqueous ammonium chloride. The organic layer was isolated, further
washed with saturated aqueous sodium bicarbonate, brine, and dried
with anhydrous sodium sulfate. The upper clear solution was
decanted and concentrated down with silica gel. The mixture was
subject to a gradient column chromatography (EtOAc-Hex 1:2 to 5:1)
to yield
3-(5-aminobenzo[b]thiophen-2-yl)-5-(1-(3-((tert-butyldimethylsilyl)oxy)pr-
opyl)-1H-tetrazol-5-yl)pyridin-2-amine as an oil, which solidified
in vacuo, in the amount of 127 mg.
[0215] .sup.1H NMR (600 MHz, DMSO-d.sub.6) .delta. ppm 8.38 (d,
J=2.05 Hz, 1H) 7.87 (d, J=2.05 Hz, 1H) 7.58 (d, J=8.66 Hz, 1H) 7.42
(s, 1H) 6.98 (d, J=1.76 Hz, 1H) 6.70-6.77 (m, 3H) 5.13 (s, 2H) 4.54
(t, J=6.97 Hz, 2H) 3.58 (t, J=5.72 Hz, 2H) 2.07 (quin, J=6.35 Hz,
2H) 0.75 (s, 9H) -0.06 (s, 6H)
[0216] To the stirring solution of
3-(5-aminobenzo[b]thiophen-2-yl)-5-(1-(3-((tert-butyldimethylsilyl)oxy)pr-
opyl)-1H-tetrazol-5-yl)pyridin-2-amine (123 mg, 0.256 mmol, 1 eq)
in anhydrous tetrahydrofuran (3 mL) under nitrogen atmosphere was
added 4-chloro-3-(trifluoromethyl)phenyl isocyanate (58 mg, 1 eq)
and the reaction mixture was stirred at room temperature for three
hours. The reaction was then diluted with ethyl acetate, washed
sequentially with aqueous ammonium chloride, saturated aqueous
sodium bicarbonate, and brine, and dried with anhydrous sodium
sulfate. The upper clear solution was decanted and concentrated
under reduced pressure and loaded onto silica. The mixture was
subject to a gradient column chromatography (EtOAc-Hex 1:5 to 2:1)
to give
1-(2-(2-amino-5-(1-(3-((tert-butyldimethylsilyl)oxy)propyl)-1H-tetrazol-5-
-yl)pyridin-3-yl)benzo[b]thiophen-5-yl)-3-(4-chloro-3-(trifluoromethyl)phe-
nyl)urea as a white solid in the amount of 130 mg.
[0217] .sup.1H NMR (600 MHz, DMSO-d.sub.6) .delta. ppm 9.19 (s, 1H)
8.99 (s, 1H) 8.41 (d, J=2.05 Hz, 1H) 8.16 (d, J=2.05 Hz, 1H) 8.14
(d, J=1.47 Hz, 1H) 7.89-7.92 (m, 2H) 7.61-7.67 (m, 3H) 7.40 (dd,
J=8.66, 1.91 Hz, 1H) 6.81 (s, 2H) 4.56 (t, J=6.90 Hz, 2H) 3.58 (t,
J=5.72 Hz, 2H) 2.08 (quin, J=6.31 Hz, 2H) 0.76 (s, 9H) -0.06 (s,
6H)
[0218] To the solution of
1-(2-(2-amino-5-(1-(3-((tert-butyldimethylsilyl)oxy)propyl)-1H-tetrazol-5-
-yl)pyridin-3-yl)benzo[b]thiophen-5-yl)-3-(4-chloro-3-(trifluoromethyl)phe-
nyl)urea (120 mg, 0.17 mmol, 1 eq) in anhydrous tetrahydrofuran (3
mL) under nitrogen atmosphere at 0.degree. C. was added dropwise a
solution of tetrabutylammonium fluoride (1.0 M in THF, 0.51 mL, 3
eq). After the reaction was stirred at room temperature for 2
hours, it was partitioned between ethyl acetate and aqueous
ammonium chloride. The organic layer was isolated, further washed
with saturated aqueous sodium bicarbonate, brine, and dried with
anhydrous sodium sulfate. The upper clear solution was decanted and
concentrated and loaded onto silica. The mixture was subject to a
gradient column chromatography (EtOAc-Hex 4:1 to MeOH-EtOAc 1:25)
to yield
1-(2-{2-amino-5-[1-(3-hydroxypropyl)-1H-tetrazol-5-yl]pyridin-3-yl}-1-ben-
zothien-5-yl)-3-[4-chloro-3-(trifluoromethyl)phenyl]urea as a white
solid in the amount of 63 mg.
[0219] .sup.1H NMR (600 MHz, DMSO-d.sub.6) .delta. ppm 9.20 (s, 1H)
9.00 (s, 1H) 8.44 (d, J=2.20 Hz, 1H) 8.16 (d, J=2.05 Hz, 1H) 8.14
(d, J=1.61 Hz, 1H) 7.96 (d, J=2.05 Hz, 1H) 7.91 (d, J=8.66 Hz, 1H)
7.68 (s, 1H) 7.61-7.67 (m, 2H) 7.40 (dd, J=8.66, 1.91 Hz, 1H) 6.81
(s, 2H) 4.69 (t, J=4.99 Hz, 1H) 4.55 (t, J=7.26 Hz, 2H) 3.44 (q,
J=5.58 Hz, 2H) 2.03 (quin, J=6.53 Hz, 2H)
##STR00033##
1-(2-{2-amino-5-[2-(2-hydroxyethyl)-2H-tetrazol-5-yl]pyridin-3-yl}-1-benz-
othien-5-yl)-3-[4-chloro-3-(trifluoromethyl)phenyl]urea
[0220] To the stirring mixture of
3-(5-aminobenzo[b]thiophen-2-yl)-5-isocyanopyridin-2-amine (1.064
g, 4 mmol, 1 eq) in anhydrous tetrahydrofuran (25 mL) under
nitrogen atmosphere was added 4-chloro-3-(trifluoromethyl)phenyl
isocyanate (904 mg, 1 eq) and the reaction mixture was stirred at
room temperature for three hours. The reaction was then diluted
with ethyl acetate, washed sequentially with aqueous ammonium
chloride, saturated aqueous sodium bicarbonate, and brine, and
dried with anhydrous sodium sulfate. The upper clear solution was
decanted and concentrated under reduced pressure to lesser amount,
to which was added a small amount of EtOAc-Hex (2:1). Upon a
filtration,
1-(2-(2-amino-5-isocyanopyridin-3-yl)benzo[b]thiophen-5-yl)-3-(4-chloro-3-
-(trifluoromethyl)phenyl)urea was obtained as white solid in the
amount of 1.37 g.
[0221] .sup.1H NMR (600 MHz, DMSO-d.sub.6) .delta. ppm 9.23 (s, 1H)
9.03 (s, 1H) 8.42 (d, J=2.05 Hz, 1H) 8.16 (d, J=2.05 Hz, 1H) 8.13
(d, J=1.76 Hz, 1H) 7.89-7.92 (m, 2H) 7.64-7.67 (m, 1H) 7.60-7.64
(m, 2H) 7.40 (dd, J=8.80, 1.76 Hz, 1H) 7.15 (br. s., 2H)
[0222] The mixture of
1-(2-(2-amino-5-isocyanopyridin-3-yl)benzo[b]thiophen-5-yl)-3-(4-chloro-3-
-(trifluoromethyl)phenyl)urea (1.3 g, 2.67 mmol, 1 eq), ammonium
chloride (286 mg, 2 eq), and sodium azide (347 mg, 2 eq) in
anhydrous dimethylformamide (8 mL) was stirred and heated to
120.degree. C. under nitrogen atmosphere for 2 hours. The mixture
was then cooled to room temperature and additional ammonium
chloride (286 mg, 2 eq) and sodium azide (347 mg, 2 eq) were added.
The reaction mixture was heated back to 120.degree. C. and stirred
at that temperature for another hour. After it was cooled to room
temperature, it was transferred into ethyl acetate and aqueous
ammonium chloride; during the process, the pH of the aqueous layer
was adjusted to .about.3.5 by using aq KHSO.sub.4 (10%). The
organic layer was isolated, washed once with saturated brine, and
dried with anhydrous sodium sulfate. The upper solution was
decanted and concentrated and loaded onto silica. Upon gradient
column chromatography (from EtOAc to MeOH-EtOAc 1:2),
1-(2-(2-amino-5-(2H-tetrazol-5-yl)pyridin-3-yl)benzo[b]thiophen-5-yl)-3-(-
4-chloro-3-(trifluoromethyl)phenyl) was obtained as a yellow solid
in the amount of 452 mg.
[0223] .sup.1H NMR (600 MHz, DMSO-d.sub.6) .delta. ppm 9.29 (s, 1H)
9.07 (s, 1H) 8.61 (s, 1H) 8.16 (br. s., 1H) 8.11 (br. s., 2H) 7.91
(d, J=8.51 Hz, 1H) 7.65-7.69 (m, 2H) 7.61-7.64 (m, 1H) 7.41 (d,
J=8.22 Hz, 1H) 6.28 (br. s., 2H)
[0224] To the mixture of
1-(2-(2-amino-5-(2H-tetrazol-5-yl)pyridin-3-yl)benzo[b]thiophen-5-yl)-3-(-
4-chloro-3-(trifluoromethyl)phenyl)urea (106 mg, 0.2 mmol, 1 eq) in
anhydrous dimethylformamide (2 mL) under nitrogen atmosphere was
added potassium carbonate (41.4 mg, 1.5 eq) and
(2-bromoethoxy)-tert-butyldimethylsilane (0.06 mL, 1.3 eq). The
resulting mixture was stirred and heated at 80.degree. C. for 2
hours. It was then diluted with ethyl acetate, washed sequentially
with aqueous ammonium chloride, saturated aqueous sodium
bicarbonate, and brine, and dried with anhydrous sodium sulfate.
The upper clear solution was decanted and concentrated under
reduced pressure with silica gel. Gradient column chromatography
(EtOAc-Hex from 1:100 to 3:1) gave
1-(2-(2-amino-5-(2-(2-((tert-butyldimethylsilyl)oxy)ethyl)-2H-tetrazol-5--
yl)pyridin-3-yl)benzo[b]thiophen-5-yl)-3-(4-chloro-3-(trifluoromethyl)phen-
yl)urea as a fluffy white solid in the amount of 77 mg.
[0225] .sup.1H NMR (600 MHz, DMSO-d.sub.6) .delta. ppm 9.21 (s, 1H)
9.01 (s, 1H) 8.68 (d, J=2.05 Hz, 1H) 8.17 (d, J=1.76 Hz, 1H) 8.13
(d, J=1.17 Hz, 1H) 8.09 (d, J=2.05 Hz, 1H) 7.91 (d, J=8.51 Hz, 1H)
7.70 (s, 1H) 7.64-7.67 (m, 1H) 7.61-7.64 (m, 1H) 7.41 (dd, J=8.51,
1.76 Hz, 1H) 6.69 (s, 2H) 4.81 (t, J=4.99 Hz, 2H) 4.13 (t, J=4.84
Hz, 2H) 0.73 (s, 9H) -0.10 (s, 6H)
[0226] To the solution of
1-(2-(2-amino-5-(2-(2-((tert-butyldimethylsilyl)oxy)ethyl)-2H-tetrazol-5--
yl)pyridin-3-yl)benzo[b]thiophen-5-yl)-3-(4-chloro-3-(trifluoromethyl)phen-
yl)urea (60 mg, 0.087 mmol, 1 eq) in anhydrous tetrahydrofuran (2
mL) under nitrogen atmosphere at 0.degree. C. was added dropwise a
solution of tetrabutylammonium fluoride (1.0 M in THF, 0.26 mL, 3
eq). After the reaction was stirred at room temperature for 2
hours, it was partitioned between ethyl acetate and aqueous
ammonium chloride. The organic layer was isolated, further washed
with saturated aqueous sodium bicarbonate, brine, and dried with
anhydrous sodium sulfate. The upper clear solution was decanted and
concentrated down with silica gel. The mixture was subject to a
gradient column chromatography (EtOAc-Hex 4:1 to MeOH-EtOAc 1:25)
to yield
1-(2-{2-amino-5-[2-(2-hydroxyethyl)-2H-tetrazol-5-yl]pyridin-3-yl}-1-benz-
othien-5-yl)-3-[4-chloro-3-(trifluoromethyl)phenyl]urea as a white
solid in the amount of 11 mg.
[0227] .sup.1H NMR (600 MHz, DMSO-d.sub.6) .delta. ppm 9.26 (br.
s., 1H) 9.06 (br. s., 1H) 8.69 (d, J=2.05 Hz, 1H) 8.17 (d, J=2.05
Hz, 1H) 8.13 (d, J=1.47 Hz, 1H) 8.10 (d, J=1.91 Hz, 1H) 7.91 (d,
J=8.66 Hz, 1H) 7.70 (s, 1H) 7.64-7.67 (m, 1H) 7.61-7.64 (m, 1H)
7.41 (dd, J=8.66, 1.76 Hz, 1H) 6.68 (s, 2H) 5.07 (t, J=5.65 Hz, 1H)
4.73 (t, J=5.21 Hz, 2H) 3.95 (q, J=5.43 Hz, 2H)
##STR00034##
3-(2-(3-((tert-butyldimethylsilyl)oxy)propyl)-2H-tetrazol-5-yl)-5-iodopyr-
idine
[0228] To the reaction mixture of 5-bromo-3-cyanopyridine (3 g,
15.9 mmol, 1 eq), sodium iodide (4.77 g, 2 eq), copper(I) iodide
(303 mg, 0.1 eq) in anhydrous 1,4-dioxane (40 mL) under nitrogen
atmosphere was added trans-N,N'-dimethylcyclohexane-1,2-diamine
(0.52 mL, 0.2 eq). After the reaction mixture was heated at
120.degree. C. for 16 hours, it was cooled to room temperature and
partitioned between aqueous ammonium chloride and ethyl acetate.
The organic layer was isolated, washed with saturated aqueous
sodium bicarbonate, brine, and dried with anhydrous sodium
chloride. The upper clear solution was decanted, concentrated, and
the brown oily residue was subject to a column chromatography
(EtOAc-Hex 1:15 to 1:4) yielding 3-iodo-5-isocyanopyridine as a
white solid in the amount of 2.41 g.
[0229] .sup.1H NMR (600 MHz, DMSO-d.sub.6) .delta. ppm 9.11 (d,
J=2.05 Hz, 1H) 9.01 (d, J=1.76 Hz, 1H) 8.78 (t, J=2.05 Hz, 1H)
[0230] To a solution of 3-iodo-5-isocyanopyridine (2.4 g, 10.43
mmol, 1 eq) in anhydrous DMF (25 mL) in a round bottom flask was
added ammonium chloride (0.95 g, 1.7 eq) and sodium azide (1.15 g,
1.7 eq). After the reaction mixture was heated and stirred at
100.degree. C. under anhydrous nitrogen atmosphere for 16 hours, it
was cooled to room temperature and poured into ice water. 2 N HCl
was dropwise added to adjust the pH .about.3 followed by a further
stirring for about 30 minutes. The aqueous layer was first
extracted with ethyl acetate, followed by an extraction with
THF-EtOAc (1:9), and lastly an extraction with i-PrOH--CHCl.sub.3
(1:6). All organic solvents were combined and dried with anhydrous
sodium sulfate. The upper clear liquor was decanted, concentrated,
and the resulting solid residue was treated with EtOAc-Hex (1:9).
After the mixture was stirred about 16 hours, it was filtered to
yield 3-iodo-5-(2H-tetrazol-5-yl)pyridine as white solid in the
amount of 2.36 g.
[0231] .sup.1H NMR (600 MHz, DMSO-d.sub.6) .delta. ppm 9.18 (d,
J=2.05 Hz, 1H) 9.01 (d, J=2.05 Hz, 1H) 8.73 (t, J=1.91 Hz, 1H)
[0232] The reaction mixture of 3-iodo-5-(2H-tetrazol-5-yl)pyridine
(273 mg, 1 mmol, 1 eq), (3-bromopropoxy)-tert-butyldimethylsilane
(0.31 mL, 1.3 eq), potassium carbonate (207 mg, 1.5 eq) in
anhydrous DMF (4 mL) under nitrogen atmosphere was stirred and
heated at 80.degree. C. for 5 hours. The mixture was then diluted
with ethyl acetate, washed sequentially with saturated aqueous
sodium bicarbonate, aqueous ammonium chloride, brine, and dried
with anhydrous sodium sulfate. The upper liquor was decanted,
concentrated under reduced pressure, and the resulting brown oily
residue was subject to a gradient column chromatography (EtOAc-Hex
1:50 to 1:4) giving
3-(2-(3-((tert-butyldimethylsilyl)oxy)propyl)-2H-tetrazol-5-yl)-5-iodopyr-
idine as a soft white solid in the amount of 395 mg.
[0233] .sup.1H NMR (600 MHz, DMSO-d.sub.6) .delta. ppm 9.18 (d,
J=1.76 Hz, 1H) 8.98 (d, J=2.05 Hz, 1H) 8.69 (t, J=2.05 Hz, 1H) 4.82
(t, J=6.60 Hz, 2H) 3.65 (t, J=5.87 Hz, 2H) 2.18 (quin, J=6.24 Hz,
2H) 0.84 (s, 9H) 0.00 (s, 6H)
##STR00035##
N-[3-({5-[2-(3-hydroxypropyl)-2H-tetrazol-5-yl]pyridin-3-yl}ethynyl)pheny-
l]-3-methyl-2-furamide
[0234] To the nitrogen bubbled solution of
3-(2-(3-((tert-butyldimethylsilyl)oxy)propyl)-2H-tetrazol-5-yl)-5-iodopyr-
idine (89 mg, 0.2 mmol, 1 eq),
N-(3-ethynylphenyl)-3-methylfuran-2-carboxamide (67.5 mg, 1.5 eq),
triphenylphosphine (1.3 mg, 0.025 eq), and triethylamine (0.09 mL,
3 eq) in anhydrous DMF (2 mL) was added
bis(triphenylphosphine)palladium(II) dichloride (14 mg, 0.1 eq),
and copper(I) iodide (8 mg, 0.2 eq). After the reaction mixture was
stirred at room temperature for 20 minutes, it was diluted with
ethyl acetate, washed sequentially with saturated aqueous sodium
bicarbonate, aqueous ammonium chloride, brine, and dried with
anhydrous sodium sulfate. The upper liquor was decanted,
concentrated under reduced pressure, and the resulting brown oily
residue was loaded onto silica and columned (EtOAc-Hex 1:20 to
1:4). The fractions containing the desired product were collected,
concentrated to a lesser amount, and the white fluffy solid was
filtered giving
N-(3-((5-(2-(3-((tert-butyldimethylsilyl)oxy)propyl)-2H-tetrazol-5-yl)pyr-
idin-3-yl)ethynyl)phenyl)-3-methylfuran-2-carboxamide in the amount
of 78 mg.
[0235] .sup.1H NMR (600 MHz, DMSO-d.sub.6) .delta. ppm 10.22 (s,
1H) 9.21 (d, J=2.05 Hz, 1H) 8.93 (d, J=1.76 Hz, 1H) 8.52 (t, J=2.05
Hz, 1H) 8.16 (t, J=1.61 Hz, 1H) 7.82 (d, J=1.47 Hz, 1H) 7.79-7.81
(m, 1H) 7.41-7.44 (m, 1H) 7.36 (dt, J=7.56, 1.21 Hz, 1H) 6.61 (d,
J=1.47 Hz, 1H) 4.84 (t, J=6.60 Hz, 2H) 3.68 (t, J=5.72 Hz, 2H) 2.36
(s, 3H) 2.20 (quin, J=6.31 Hz, 2H) 0.85 (s, 9H) 0.02 (s, 6H)
[0236] To the solution of
N-(3-((5-(2-(3-((tert-butyldimethylsilyl)oxy)propyl)-2H-tetrazol-5-yl)pyr-
idin-3-yl)ethynyl)phenyl)-3-methylfuran-2-carboxamide (71 mg, 0.131
mmol, 1 eq) in anhydrous THF (2.6 mL) under nitrogen atmosphere at
0.degree. C. was added dropwise a solution of tetrabutylammonium
fluoride (1.0 M in THF, 0.393 mL, 3 eq). The clear reaction
solution was stirred at 0.degree. C. using an ice-bath for 3 hours.
The solution was then partitioned between ethyl acetate and
saturated aqueous sodium bicarbonate. The organic layer was
isolated, washed with aqueous ammonium chloride, brine, and dried
with anhydrous sodium sulfate. The upper clear liquor was decanted,
concentrated, and the oily residue was loaded onto silica and
chromatographed (EtOAc-Hex 1:4 to 4:1) to yield
N-[3-({5-[2-(3-hydroxypropyl)-2H-tetrazol-5-yl]pyridin-3-yl}ethynyl)pheny-
l]-3-methyl-2-furamide as a white solid in the amount of 28 mg.
[0237] .sup.1H NMR (600 MHz, DMSO-d.sub.6) .delta. ppm 10.22 (s,
1H) 9.22 (d, J=2.05 Hz, 1H) 8.94 (d, J=2.05 Hz, 1H) 8.53 (t, J=2.05
Hz, 1H) 8.16 (t, J=1.76 Hz, 1H) 7.82 (d, J=1.47 Hz, 1H) 7.79-7.82
(m, 1H) 7.41-7.44 (m, 1H) 7.37 (dt, J=7.63, 1.17 Hz, 1H) 6.61 (d,
J=1.47 Hz, 1H) 4.84 (t, J=7.04 Hz, 2H) 4.72 (t, J=4.99 Hz, 1H) 3.49
(q, J=5.87 Hz, 2H) 2.36 (s, 3H) 2.15 (quin, J=6.60 Hz, 2H)
##STR00036##
N-[3-({5-[2-(3-hydroxypropyl)-2H-tetrazol-5-yl]pyridin-3-yl}ethynyl)pheny-
l]-3-methylbenzamide
[0238] Synthesized using a procedure similar to the one used for
the synthesis of Compound 9.
[0239] .sup.1H NMR (600 MHz, DMSO-d.sub.6) .delta. ppm 10.35 (s,
1H) 9.22 (d, J=2.05 Hz, 1H) 8.94 (d, J=1.76 Hz, 1H) 8.54 (t, J=2.05
Hz, 1H) 8.14 (t, J=1.76 Hz, 1H) 7.82-7.84 (m, 1H) 7.79 (s, 1H)
7.75-7.78 (m, 1H) 7.41-7.48 (m, 3H) 7.39 (dt, J=7.63, 1.17 Hz, 1H)
4.84 (t, J=7.04 Hz, 2H) 4.72 (t, J=4.99 Hz, 1H) 3.49 (q, J=5.87 Hz,
2H) 2.41 (s, 3H) 2.15 (quin, J=6.53 Hz, 2H)
##STR00037##
1-[3-({2-amino-5-[2-(3-hydroxypropyl)-2H-tetrazol-5-yl]pyridin-3-yl}ethyn-
yl)phenyl]-3-[4-chloro-3-(trifluoromethyl)phenyl]urea
[0240] A mixture of
3-((3-aminophenyl)ethynyl)-5-(2-(3-((tert-butyldimethylsilyl)oxy)propyl)--
2H-tetrazol-5-yl)pyridin-2-amine (135 mg, 0.3 mmol, 1 eq) and
4-chloro-3-(trifluoromethyl)phenyl isocyanate (67 mg, 1 eq) in
anhydrous tetrahydrofuran (3 mL) was stirred at room temperature
and under nitrogen atmosphere for 3 hours. The mixture was then
diluted with ethyl acetate, washed sequentially with aqueous
ammonium chloride, saturated aqueous sodium bicarbonate, and brine,
and dried with anhydrous sodium sulfate. The clear solution was
decanted, concentrated under reduced pressure, and the resulting
clear oil was dried in vacuo for 5 minutes. It was then treated
with a small amount of EtOAc-Hex (1:9), followed by stirring at
room temperature for 30 minutes. The white solid that appeared
during the process was filtered giving
1-(3-((2-amino-5-(2-(3-((tert-butyldimethylsilyl)oxy)propyl)-2H-tetrazol--
5-yl)pyridin-3-yl)ethynyl)phenyl)-3-(4-chloro-3-(trifluoromethyl)phenyl)ur-
ea in the amount of 201 mg.
[0241] .sup.1H NMR (600 MHz, DMSO-d.sub.6) .delta. ppm 9.25 (s, 1H)
8.95 (s, 1H) 8.63 (s, 1H) 8.14 (s, 2H) 7.83 (s, 1H) 7.60-7.67 (m,
2H) 7.44 (br. s., 1H) 7.36 (d, J=4.11 Hz, 2H) 6.92 (br. s., 2H)
4.75 (t, J=6.60 Hz, 2H) 3.65 (t, J=5.72 Hz, 2H) 2.16 (quin, J=6.16
Hz, 2H) 0.85 (s, 9H) 0.02 (s, 6H)
[0242] To the solution of
1-(3-((2-amino-5-(2-(3-((tert-butyldimethylsilyl)oxy)propyl)-2H-tetrazol--
5-yl)pyridin-3-yl)ethynyl)phenyl)-3-(4-chloro-3-(trifluoromethyl)phenyl)ur-
ea (188 mg, 0.28 mmol, 1 eq) in anhydrous tetrahydrofuran (5 mL)
under nitrogen atmosphere at 0.degree. C. was added dropwise
tetrabutylammonium fluoride (1.0 M in THF, 0.84 mL, 3 eq). The
reaction was stirred at room temperature for 3 hours and then
concentrated and loaded onto silica. The mixture was subject to a
column chromatography (EtOAc-Hex 4:1 to MeOH-EtOAc 1:25) to yield
1-[3-({2-amino-5-[2-(3-hydroxypropyl)-2H-tetrazol-5-yl]pyridin-3-yl}ethyn-
yl)phenyl]-3-[4-chloro-3-(trifluoromethyl)phenyl]urea as a white
solid in the amount of 127 mg.
[0243] .sup.1H NMR (600 MHz, DMSO-d.sub.6) .delta. ppm 9.26 (s, 1H)
8.96 (s, 1H) 8.64 (s, 1H) 8.14 (d, J=7.04 Hz, 2H) 7.82 (s, 1H)
7.61-7.67 (m, 2H) 7.43-7.46 (m, 1H) 7.34-7.37 (m, 2H) 6.91 (br. s.,
2H) 4.75 (t, J=7.04 Hz, 2H) 4.70 (t, J=4.99 Hz, 1H) 3.47 (q, J=5.58
Hz, 2H) 2.11 (quin, J=6.46 Hz, 2H)
##STR00038##
1-[2-fluoro-5-(trifluoromethyl)phenyl]-3-[4-({5-[2-(3-hydroxypropyl)-2H-t-
etrazol-5-yl]pyridin-3-yl}ethynyl)phenyl]urea
[0244] Synthesized using a procedure similar to the one used for
the synthesis of Compound 11. .sup.1H NMR (600 MHz, DMSO-d.sub.6)
.delta. ppm 9.43 (s, 1H) 9.19 (d, J=1.47 Hz, 1H) 8.98 (d, J=2.05
Hz, 1H) 8.89 (d, J=1.47 Hz, 1H) 8.61 (dd, J=7.04, 1.47 Hz, 1H) 8.49
(s, 1H) 7.56-7.61 (m, 4H) 7.51 (dd, J=10.12, 9.24 Hz, 1H) 7.42 (dt,
J=7.92, 3.67 Hz, 1H) 4.84 (t, J=7.04 Hz, 2H) 4.72 (t, J=4.99 Hz,
1H) 3.49 (q, J=5.87 Hz, 2H) 2.15 (quin, J=6.53 Hz, 2H).
##STR00039##
1-[2-(2-amino-5-pyrimidin-2-ylpyridin-3-yl)-1-benzothien-5-yl]-3-[4-chlor-
o-3-(trifluoromethyl)phenyl]urea
[0245]
1-{2-[2-amino-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridi-
n-3-yl]-1-benzothien-5-yl}-3-[4-chloro-3-(trifluoromethyl)phenyl]urea
(194 mg, 0.33 mmoles) and 2-chloropyrimidine (38 mg, 0.3 mmoles)
was added to a mixture of 6 ml of dioxane and 2 ml of 2M aqueous
Sodium Carbonate. Next, Palladium(II) Acetate (10 mol %, 7 mg) and
Triphenylphosphene (20 mol %, 16 mg) was added, followed by 2 ml of
dioxane. Dry nitrogen was bubbled through the resulting solution
for 15 minutes. Following this, the reaction mixture was set up
with a reflux condenser, under nitrogen atmosphere, and heated at
95.degree. C. overnight. The reaction was then cooled to room
temperature and 40 ml of ethyl acetate was added. The mixture was
transferred to a separatory funnel and extracted with saturated
Sodium Bicarbonate (3.times.40 ml) followed by saturated NaCl
(3.times.40 ml). The organic layer was dried with anhydrous Sodium
Sulfate, loaded onto silica and columned using ethyl
acetate/hexanes, to give 30 mg of the product.
[0246] .sup.1H NMR (<dmso>) .delta.: 9.19 (s, 1H), 9.02 (d,
J=2.1 Hz, 1H), 8.99 (s, 1H), 8.83 (d, J=4.7 Hz, 2H), 8.47 (d, J=2.1
Hz, 1H), 8.14 (dd, J=13.8, 2.1 Hz, 2H), 7.90 (d, J=8.8 Hz, 1H),
7.68 (s, 1H), 7.58-7.66 (m, 2H), 7.41 (dd, J=8.8, 2.1 Hz, 1H), 7.35
(t, J=4.8 Hz, 1H), 6.71 (s, 2H).
##STR00040##
1-[2-(2-amino-5-pyrazin-2-ylpyridin-3-yl)-1-benzothien-5-yl]-3-[4-chloro--
3-(trifluoromethyl)phenyl]urea
[0247] Synthesized using a procedure similar to Example 13.
[0248] .sup.1H NMR (<dmso>) .delta.: 9.24 (d, J=1.5 Hz, 1H),
9.20-9.23 (m, 1H), 9.00 (s, 1H), 8.83 (d, J=2.3 Hz, 1H), 8.64 (dd,
J=2.5, 1.6 Hz, 1H), 8.51 (d, J=2.3 Hz, 1H), 8.30 (d, J=2.3 Hz, 1H),
8.16 (d, J=2.3 Hz, 1H), 8.12 (d, J=2.1 Hz, 1H), 7.91 (d, J=8.8 Hz,
1H), 7.59-7.72 (m, 3H), 7.41 (dd, J=8.6, 2.2 Hz, 1H), 6.61 (s,
2H).
[0249] Biological data for the compounds of the present invention
was generated by use of the following assays.
VEGFR2 Kinase Assay
[0250] Biochemical KDR kinase assays were performed in 96 well
microtiter plates that were coated overnight with 75 .mu.g/well of
poly-Glu-Tyr (4:1) in 10 mM Phosphate Buffered Saline (PBS), pH
7.4. The coated plates were washed with 2 mls per well PBS +0.05%
Tween-20 (PBS-T), blocked by incubation with PBS containing 1% BSA,
then washed with 2 mls per well PBS-T prior to starting the
reaction. Reactions were carried out in 100 .mu.L reaction volumes
containing 2.7 .mu.M ATP in kinase buffer (50 mM Hepes buffer pH
7.4, 20 mM MgCl.sub.2, 0.1 mM MnCl.sub.2 and 0.2 mM
Na.sub.3VO.sub.4). Test compounds were reconstituted in 100% DMSO
and added to the reaction to give a final DMSO concentration of 5%.
Reactions were initiated by the addition 20 ul per well of kinase
buffer containing 200-300 ng purified cytoplasmic domain KDR
protein (BPS Bioscience, San Diego, Calif.). Following a 15 minute
incubation at 30.degree. C., the reactions were washed 2 mls per
well PBS-T. 100 .mu.l of a monoclonal anti-phosphotyrosine
antibody-peroxidase conjugate diluted 1:10,000 in PBS-T was added
to the wells for 30 minutes. Following a 2 mls per well wash with
PBS-Tween-20, 100 .mu.l of O-Phenylenediamine Dihydrochloride in
phosphate-citrate buffer, containing urea hydrogen peroxide, was
added to the wells for 7-10 minutes as a colorimetric substrate for
the peroxidase. The reaction was terminated by the addition of 100
.mu.l of 2.5N H.sub.2SO.sub.4 to each well and read using a
microplate ELISA reader set at 492 nm. IC.sub.50 values for
compound inhibition were calculated directly from graphs of optical
density (arbitrary units) versus compound concentration following
subtraction of blank values.
PDGFR.beta. Kinase Assay
[0251] Biochemical PDGFR.beta. kinase assays were performed in 96
well microtiter plates that were coated overnight with 75 .mu.g of
poly-Glu-Tyr (4:1) in 10 mM Phosphate Buffered Saline (PBS), pH
7.4. The coated plates were washed with 2 mls per well PBS+0.05%
Tween-20 (PBS-T), blocked by incubation with PBS containing 1% BSA,
then washed with 2 mls per well PBS-T prior to starting the
reaction. Reactions were carried out in 100 .mu.L reaction volumes
containing 36 .mu.M ATP in kinase buffer (50 mM Hepes buffer pH
7.4, 20 mM MgCl.sub.2, 0.1 mM MnCl.sub.2 and 0.2 mM
Na.sub.3VO.sub.4). Test compounds were reconstituted in 100% DMSO
and added to the reaction to give a final DMSO concentration of 5%.
Reactions were initiated by the addition 20 ul per well of kinase
buffer containing 200-300 ng purified cytoplasmic domain PDGFR-b
protein (Millipore). Following a 60 minute incubation at
300.degree. C., the reactions were washed 2 mls per well PBS-T. 100
.mu.l of a monoclonal anti-phosphotyrosine antibody-peroxidase
conjugate diluted 1:10,000 in PBS-T was added to the wells for 30
minutes. Following a 2 mls per well wash with PBS-Tween-20, 100
.mu.l of O-Phenylenediamine Dihydrochloride in phosphate-citrate
buffer, containing urea hydrogen peroxide, was added to the wells
for 7-10 minutes as a colorimetric substrate for the peroxidase.
The reaction was terminated by the addition of 100 .mu.l of 2.5N
H.sub.2SO.sub.4 to each well and read using a microplate ELISA
reader set at 492 nm. IC.sub.50 values for compound inhibition were
calculated directly from graphs of optical density (arbitrary
units) versus compound concentration following subtraction of blank
values.
PKR KinaseGlo Assay
[0252] Commercially available recombinant human GST-PKR
(SignalChem, Canada; 1.5 uM-2 uM stock) is diluted to 500 nM in
assay buffer (20 mM Tris-HCl, pH 7.2, 10 mM KCl, 10 mM MgCl2, 10%
glycerol). Preactivated PKR is dispensed to 384/96-well black
plates at 3.125/12.5 uls/well using the liquid handler Janus.
Appropriate dilutions of inhibitors are added to 384/96-well plate
followed by 6.6 uM ATP (final) and incubated for 10 minutes at room
temperature. The remaining ATP/well is determined by adding 6.25/25
uls/well Kinase-Glo assay mix (Promega) and luminescence is
measured on EnVision luminescence plate reader (integration time,
0.2 sec; Perkin-Elmer, Mass., USA). The % inhibition for the
compounds is calculated using ATP only (100% inhibition) and
PKR+ATP (0% inhibition). IC50 values are determined by plotting %
activity versus inhibitor concentration. Curves are fitted using
Activity base XLfit (IDBS, UK) using the formula-- [0253] 4
Parameter Logistic Model
[0253] fit=(A+((B-A)/(1+(10 ((C-x)*D)))))
inv=(C-(log(((B-A)/(y-A))-1)/D))
res=(y-fit)
[0254] The biological results for the various compounds are shown
in Table 1 below.
TABLE-US-00002 TABLE 1 In vitro VEGFR2 and PDGFR.beta. data VEGFR2
PKR PDGFR.beta. Kinase KinaseGlo Kinase Assay Assay Assay Ex.
Structure (IC.sub.50 nM) (IC.sub.50 nM) (IC.sub.50 nM) 1
##STR00041## na 727 na 2 ##STR00042## na 140 na 3 ##STR00043## na
39 na 4 ##STR00044## na 99 na 5 ##STR00045## na 76 na 6
##STR00046## na 66 na 7 ##STR00047## na na na 8 ##STR00048## na 82
na 9 ##STR00049## 5 >10,000 21 10 ##STR00050## 7 na na 11
##STR00051## na 158 na 12 ##STR00052## 8 >10,000 na 13
##STR00053## na 558 na 14 ##STR00054## na 301 na
* * * * *